WO2009035955A1 - Methods for detecting enterobacter sakazakii - Google Patents

Abstract

The present invention provides methods and oligonucleotides for detecting Enterobacter sakazakii in a sample, wherein the specific target sequence is within the mms operon.

Description

METHODS FOR DETECTING ENTEROBACTER SAKAZAKII

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No. 60/971,680, filed September 12, 2007.

BACKGROUND

Enterobacter sakazakii is a bacterium belonging to the family Enterobacteriaceae, which contains a number of bacterial species that are commonly found among the intestinal flora in humans and animals and may be found in the environment. The World Health Organization has reported that E. sakazakii has been implicated in outbreaks causing meningitis or enteritis, some of which have resulted in particularly high mortality among infants that were infected with the organism. The infections can also result in severe, lasting complications that may include neurological disorders.

Although E. sakazakii has been detected in other types of food, powdered infant formula has been linked specifically to outbreaks of disease. The organism can enter the powdered infant formula during production and is capable of surviving process and storage conditions. Additionally, the organisms can be introduced into the infant formula as it is being reconstituted by the user.

E. sakazakii was described as a bacterial species in 1980. It was formerly known as yellow pigmented Enterobacter cloacae. As reported by Leuschner, Baird, Donald and Cox in "A Medium for the Presumptive Detection of E. sakazakii in Infant Formula," Food Microbiology 21 (2004), 527-533, E. sakazakii has been implicated in a severe form of neonatal meningitis with a high mortality rate. It is reported that many newborns with E. sakazakii meningitis die within days of infection, and that the case- fatality rates vary between 40 and 80%, Nazarowec-White and Farber, "E. sakazakii: A Review, International Journal of Food Microbiology 34 (1997) 103-113. While a reservoir for E. sakazakii bacteria is unknown, reports have suggested that powdered milk-based infant formula may be a vehicle for infection. There have also been reported cases of infection in adults caused by E. sakazakii bacteria.

Accordingly, there is a clear need for a rapid and accurate device for detecting and identifying E. sakazakii bacteria in food, patient samples, and on surfaces. Researchers have reported the use of nutrient agar plating media that is responsive to the alpha-glucosidase enzyme, but such media are subject to the production of false negatives, have been time consuming, and produce plates that are difficult to read and analyze because of colonies of unwanted microorganisms. Accordingly, such media have serious drawbacks for isolating and enumerating E. sakazakii from foods or the diagnosis of infections in newborns and adults. The article by Leuschner, Baird, Donald and Cox cited above describes the detection and identification of E. sakazakii in infant formula using a nutrient agar supplemented with the enzyme substrate 4-methyl- umbelliferyl-alpha-D-glucoside. This plating medium will produce a substantial number of false negatives, because some E. sakazakii isolates can not utilize the substrate 4-methyl-umbelliferyl-alpha-D-glucoside. Further, the detection and identification process was excessively time consuming, requiring separate enrichment and testing steps. Traditional detection methods based on the cultivation of the organisms on agar media typically require relatively long incubation periods and the results may be inconclusive, due to poor selectivity or differentiation of mixed cultures on the plating media.

SUMMARY OF THE INVENTION

There is a need for a nucleic acid-based detection method to provide a more rapid, specific and sensitive test than the traditional culture methods.

The present invention includes methods for detecting E. sakazakii in a biological sample. For instance, the method may include amplifying a target polynucleotide present in a biological sample to result in an amplified product, wherein the target polynucleotide is associated with Enterobacter sakazakii. The target polynucleotide may be a mms polynucleotide from the mms

(macromolecular synthesis) operon, for instance, a polynucleotide including SEQ ID NO:3, or a portion thereof. Examples of primers that can be used to amplify such a polynucleotide include, for instance, a first primer that includes a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, and a second primer that includes a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186-235 of SEQ ID NO:3.

In one aspect, the methods may include amplifying a target polynucleotide present in a biological sample to result in an amplified product, wherein the biological sample is contacted with a first mms primer and a second mms primer under suitable conditions to result in an amplified product. The first mms primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, and the second mms primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3.

The amplified product is detected, wherein the presence of the amplified product is indicative of the presence of E. sakazakii in the biological sample.

In another aspect, the methods may include contacting a biological sample with a first mms primer and a second mms primer to form a mixture. The first mms primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, and the second mms primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3. The mixture is exposed to conditions suitable to form an amplified product if a mms polynucleotide is present in the biological sample, and the absence of the amplified product is detected, wherein the absence of the amplified product is indicative of the absence of E. sakazakii the biological sample.

In another aspect, the methods of the present invention may further include obtaining a biological sample. The biological sample may be from an individual suspected of infection with E. sakazakii, and the biological sample may be obtained from fecal material. The biological sample may be obtained from a food or a beverage for consumption or from a raw material that is used to produce food or beverages for consumption. The biological sample may come from a food processing environment, food processing equipment, or from containers that are used for temporary storage of food or beverages or to store materials used to make food or beverages. The detecting of the presence or absence of an amplified product may be performed after each cycling step.

In another aspect, the present invention also provides methods for isolating a polynucleotide. The methods may include providing a mixture of single stranded polynucleotides, exposing the mixture to an oligonucleotide under conditions suitable for specific hybridization of the oligonucleotide to a single stranded polynucleotide to result in a hybrid. The oligonucleotide includes a nucleotide sequence selected from one having at least about 80% identity to SEQ ID NO: 1, at least about 80% identity to SEQ ID NO:2. The hybrid may then be washed to remove contaminants. The oligonucleotide may include an affinity label, and the oligonucleotide may be attached to a solid phase material before or after the exposing. The mixture may be obtained from a biological sample, and the method can further include denaturing the polynucleotides present in the biological sample to result in single stranded polynucleotides.

Also included in the present invention are kits. The kit can include packaging materials, a first mms primer, a second mms primer. The first primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:1 and the second primer may include a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3.

In another aspect, the present invention includes an isolated polynucleotide wherein the polynucleotide is a product of DNA amplification and wherein the polynucleotide is about 50 base pairs in length. The 50 base pair polynucleotide may have at least about 80% identity to SEQ ID NO:3. In another aspect, the present invention includes an isolated first polynucleotide wherein the first polynucleotide comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, wherein the first polynucleotide amplifies a second polynucleotide comprising nucleotides 186 - 235 of SEQ ID NO:3 when used with SEQ ID NO:2. In another aspect, the present invention includes an isolated first polynucleotide wherein the first polynucleotide comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the first polynucleotide amplifies a second polynucleotide comprising nucleotides 186 - 235 of SEQ ID NO:3 when used with SEQ ID NO:2. In another aspect, the present invention includes a solid support comprising an amplified polynucleotide with at least about 80% identity to nucleotides 186 - 235 of SEQ ID NO:3.

Definitions As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxynucleotides, or peptide nucleic acids (PNA), and includes both double- and single-stranded RNA, DNA, and PNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding regions, and non-coding regions such as regulatory regions. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. An "oligonucleotide" refers to a polynucleotide of the present invention, typically a primer and/or a probe.

A "target polynucleotide," as used herein, contains a polynucleotide sequence of interest, for which amplification is desired. The target sequence may be known or not known, in terms of its actual sequence. A "coding region" is a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term "operably linked" refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

"Primer," as used herein, is an oligonucleotide that is complementary to at least a portion of target polynucleotide and, after hybridization to the target polynucleotide, may serve as a starting-point for an amplification reaction and the synthesis of an amplification product. A "primer pair" refers to two primers that can be used together for an amplification reaction, "mms primers" refers to a primer pair that hybridizes to mms polynucleotides, and can initiate amplification under the appropriate conditions.

The terms "complement" and "complementary" as used herein, refer to the ability of two single stranded polynucleotides (for instance, a primer and a target polynucleotide) to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5'- ATGC and 5'-GCAT are complementary. The terms "substantial complement" and "substantially complementary" as used herein, refer to a polynucleotide that is capable of selectively hybridizing to a specified polynucleotide under stringent hybridization conditions. Stringent hybridization can take place under a number of pH, salt and temperature conditions. The pH can vary from 6 to 9, preferably 6.8 to 8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium. The temperature of the hybridization reaction can vary from 30⁰C to 80⁰C, preferably between 45°C and 70⁰C. Additionally, other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as at or approaching room temperature. Among the compounds contemplated for lowering the temperature requirements is formamide. Thus, a polynucleotide is typically "substantially complementary" to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide. As used herein, "specific hybridization" refers to hybridization between two polynucleotides under stringent hybridization conditions.

"Identity" refers to sequence similarity between an oligonucleotide, such as a primer, and at least a portion of a target polynucleotide or an amplification product.

The similarity is determined by aligning the residues of the two polynucleotides (i.e., the nucleotide sequence of a primer and a reference nucleotide sequence) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least about 80% identity, at least about 85% identity, at least about 90% identity, or at least about 95% identity. Sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wisconsin), MacVector 4.5 (Kodak/IBI software package) or other suitable sequencing programs or methods known in the art. Preferably, sequence similarity between a primer and a target polynucleotide, or between a probe and an amplification product is determined using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match = 1 , penalty for mismatch = -2, open gap penalty = 5, extension gap penalty = 2, gap x dropoff = 50, expect = 10, wordsize = 11, and optionally, filter on. In the comparison of two nucleotide sequences using the BLAST search algorithm, sequence similarity is referred to as "identities."

A "label" refers to a moiety attached (covalently or non-covalently), or capable of being attached, to an oligonucleotide, which provides or is capable of providing information about the oligonucleotide (e.g., descriptive or identifying information about the oligonucleotide) or another polynucleotide with which the labeled oligonucleotide interacts (e.g., hybridizes). Labels can be used to provide a detectable (and optionally quantifiable) signal. Labels can also be used to attach an oligonucleotide to a surface. A "fluorophore" is a moiety that can emit light of a particular wavelength following absorbance of light of shorter wavelength. The wavelength of the light emitted by a particular fluorophore is characteristic of that fluorophore. Thus, a particular fluorophore can be detected by detecting light of an appropriate wavelength following excitation of the fluorophore with light of shorter wavelength. The term "quencher" as used herein refers to a moiety that absorbs energy emitted from a fluorophore, or otherwise interferes with the ability of the fluorescent dye to emit light. A quencher can re-emit the energy absorbed from a fluorophore in a signal characteristic for that quencher, and thus a quencher can also act as a fluorophore (a fluorescent quencher). This phenomenon is generally known as fluorescent resonance energy transfer (FRET). Alternatively, a quencher can dissipate the energy absorbed from a fluorophore as heat (a non-fluorescent quencher).

A "biological sample" refers to a sample obtained from eukaryotic or prokaryotic sources. Examples of eukaryotic sources include mammals, such as a human (e.g. a patient). Examples of prokaryotic sources include bacteria, such as E. sakazakii. The biological sample can be, for instance, in the form of a single cell, in the form of a tissue, or in the form of a fluid. Cells or tissue can be derived from in vitro culture. Biological samples also include samples of food, food ingredients, food residue, beverages, beverage ingredients, or beverage residue, samples from process equipment, and water (e.g., potable water or process water).

Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as hybridization, strand extension, and the like, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Such conditions, known in the art and described herein, may depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. These conditions may also depend on what event is desired, such as hybridization, cleavage, strand extension or transcription.

An "isolated" polynucleotide refers to a polynucleotide that has been removed from its natural environment. A "purified" polynucleotide is one that is at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated. The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements. The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes methods for detecting polynucleotides that are characteristic of a target nucleic acid sequence, such as a target nucleic acid sequence from E. sakazakii. For instance, the present invention includes methods directed to detecting a portion of a mms coding region present in E. sakazakii using amplification techniques and oligonucleotides, such as primers and probes. Using the methods of the present invention, it is possible to identify the presence of E. sakazakii in a biological sample. The present invention also includes the oligonucleotides described herein. The present invention also includes amplifying and detecting small (e.g., about 50 base pair) oligonucleotide sequences. The amplification of such small target nucleotide sequences affords the advantage of very short periods for primer extension during the polymerase chain reaction (PCR) assays, thus enabling a faster assay.

Oligonucleotides

Oligonucleotides of the present invention include primers that can be used to amplify a portion of a mms coding region. Alternatively, the oligonucleotides may be used in a simple elongation reaction, when mixed with a solution containing an appropriate buffer, polymerase enzyme, and deoxyribonucleotide triphosphates and annealed to a suitable complementary template. An example of a mms coding region is disclosed at SEQ ID NO:3 (Genbank accession number LO 1755). Primers useful for amplifying a portion of a mms coding region may amplify a region of SEQ ID NO:3, preferably a region that includes nucleotides from about 186 to about 235 of SEQ ID NO:3. Accordingly, the nucleotide sequence of a primer may correspond to nucleotides from about 186 to about 208, preferably nucleotides 186 to 208 (referred to herein as SEQ ID NO: 1). Likewise, the nucleotide sequence of a primer may correspond to the complement of nucleotides from about 211 to about 235, preferably 211 to 235

(referred to herein as SEQ ID NO:2). Examples of primer pairs useful to amplify a portion of a mms coding region include, but are not limited to, the following: SEQ ID NO:1 and SEQ ID NO:2; a primer having sequence similarity to SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:1 and a primer having sequence similarity to SEQ ID NO:2; and a primer having sequence similarity to SEQ ID NO: 1 and a primer having sequence similarity to SEQ ID NO:2. Typically, primers for PCR amplification are about 20-25 nucleotides in length and amplify a polynucleotide of about 100-150 nucleotides or more in length. The proximal terminal nucleotides of the inventive primers of the present disclosure are separated by a very short distance (e.g. 2 nucleotides). The primers of SEQ ID NO:1 and SEQ ID NO:2 were selected to be in close proximity to one another in order to minimize the time necessary for primer extension in a PCR assay. Primers that amplify a mms coding region can be designed using readily available computer programs, such as Primer Express® (Applied Biosystems, Foster City, CA), and IDT® OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, IA). Factors that can be considered in designing primers include, but are not limited to, melting temperatures, primer length, size of the amplification product, and specificity. Primers useful in the amplification methods described herein typically have a melting temperature (T_M) that is greater than at least 56°C, at least 57°C, at least 58°C, or at least 59°C. The T_M of a primer can be approximated by the Wallace Rule (Wallace et al, 1979, Nucleic Acids Res., 6:3543-3557) or by using a computer program, such as IDT Oligo Analyzer 3.0. Typically, the primers of a primer pair will have T_MS that vary by no greater than 4°C, no greater than 3°C, no greater than 2°C, or no greater than 1 ⁰C. Typically, two primers are long enough to hybridize to the target polynucleotide and not hybridize to other non-target polynucleotides present in microbes, preferably, other members of the family Enterobacteriaceae, other species of Enterobacter, and other polynucleotides that may be present in the amplification reaction. Primer length is generally between about 15 and about 30 nucleotides (for instance, 15, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides).

A primer useful in the present invention may have sequence similarity to SEQ ID NO: 1 and SEQ ID NO:2. Non-complementary nucleotides in such a primer with sequence similarity can be located essentially anywhere throughout the primer. In some aspects, it is preferable to preserve cytosine or guanine residues. For instance, in a primer with sequence similarity to SEQ ID NO:1, it is more preferable to alter one or more adenine or thymine residues in SEQ ID NO:1, and preserve the cytosine and guanine residues. Preferably, the first nucleotide at the 3' end of a primer with sequence similarity is identical to the corresponding first nucleotide in SEQ ID NO:1 and SEQ ID NO:2. A primer having sequence similarity to SEQ ID NO:1 and SEQ ID NO:2 has the activity of amplifying a target polynucleotide under the appropriate conditions. Whether such a candidate primer (i.e., a primer being compared to SEQ ID NO:1 or SEQ ID NO:2) having sequence similarity has the activity of amplifying a target polynucleotide can be tested using a Mastercycler Personal (Eppendorf, Germany) thermal cycler with the following profile: 94°C for 4 minutes, then 40 cycles of 94°C for 20 seconds, 58°C for 20 seconds, and 72 degrees for 60 seconds. Optionally, a final extension step (e.g., for up to several minutes at 72 degrees) may be added to complete the last round of DNA synthesis. Amplification can be performed with the two primers using readily available kits (including Master Mix and Taq polymerase) for PCR reactions and following the manufacturer's instructions provided therein. The target polynucleotide for evaluating a candidate primer having sequence similarity to either SEQ ID NO:1 or SEQ ID NO:2 is one that includes nucleotides 186 to 235 of SEQ ID NO:3. Such a nucleotide sequence is present in whole cell DNA obtained from the E. sakazakii designated Tecra International Culture Collection strain number 4217. When testing a candidate primer having sequence similarity to SEQ ID NO:1, the second primer used is SEQ ID NO:2. When testing a candidate primer having sequence similarity to SEQ ID NO:2, the second primer used is SEQ ID NO:1. It is anticipated that oligonucleotides of the present invention may be used with appropriate probes that can hybridize to at least a portion of an amplified product that results from the use ofmms primers. Such probes, which may be useful in performing real-time PCR, can be selected according to general principles known in the art for PCR probe selection. Factors that can be considered in designing probes useful in the real-time PCR methods include, but are not limited to, melting temperature, length, location of the probe with respect to the primers. Typically, a probe will have a T_M that is greater than the highest T_M of the primers with which the probe is to be used. Preferably, a probe has a T_M that is at least 5°C greater, at least 6°C greater, at least 7°C greater, at least 8°C greater, at least 8.5°C greater, at least 9°C greater, or at least 9.5°C greater than the highest T_M of the primer pair with which the probe is to be used.

Typically, the greater Tm permits the probe to hybridize before the primer, which aids in maximizing the labeling of each amplification product with probe. Typically, a probe is long enough to hybridize to the target polynucleotide (and the amplification product) and not hybridize to other non-target polynucleotides present in a microbe, and other polynucleotides that may be present in the amplification reaction. Probe lengths are generally between about 15 nucleotides and about 30 nucleotides. Preferably, a probe and the primers with which the probe is used will not hybridize to the same nucleotides of an amplification product. A probe will hybridize to one strand of an amplified product, and is typically designed to hybridize to the amplified product before the primer that hybridizes to that strand. In some aspects of the present invention, a probe hybridizes to one strand of an amplified product within no more than 1, 2, 3, 4, or 5 nucleotides of the primer that hybridizes to the same strand. In some aspects of the invention that involve the use of two probes, the two probes preferably hybridize to the same strand of an amplified product, and the two probes may optionally hybridize to the same amplification product within 1, 2, 3, 4, or 5 nucleotides of each other. A primer of the present invention may further include additional nucleotides.

Typically, such additional nucleotides are present at the 5' end of the primer, and include, for instance, nucleotides that include a restriction endonuclease site, nucleotides that form a hairpin loop, and other nucleotides that permit the primer to be used as, for instance, a scorpions primer (see, for instance, Whitcombe et al., U.S. Patent 6,326,145, and Whitcombe et al., Nat. BiotechnoL, 1999;17:804-817), an amplifluor primer (see, for instance, Nazarenko et al., Proc. Natl. Acad. Sci. USA, 1997;25:2516-2521), or a method using anti-primer quenching based real-time PCR (Li et al., Clinical Chem., 2006; 52(4):624-633). When a primer includes such additional nucleotides, the additional nucleotides are not included when determining if the primer has sequence similarity to SEQ ID NO:1 or SEQ ID NO:2. Likewise, the additional nucleotides are not included in determining the length of a primer, which is generally between about 10 and about 50 nucleotides.

Oligonucleotides of the present invention include probes that can be used to hybridize to at least a portion of an amplified product that results from the use of mms primers. Such mms probes useful herein hybridize to a region that includes nucleotides from about 186 to about 235 of SEQ ID NO:3, preferably nucleotides 186 to 235 of SEQ ID NO:3. Nucleotides of an oligonucleotide of the present invention may be modified. Such modifications can be useful to increase stability of the polynucleotide in certain environments. Modifications can include a nucleic acid backbone, base, sugar, or any combination thereof. The modifications can be synthetic, naturally occurring, or non- naturally occurring. A polynucleotide of the present invention can include modifications at one or more of the nucleic acids present in the polynucleotide. Examples of backbone modifications include, but are not limited to, phosphonoacetates, thiophosphonoacetates, phosphorothioates, phosphorodithioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (Nielson et al, U.S. Pat. No. 5,539,082;

Egholm et al., Nature, 1993, 365:566-568). Examples of nucleic acid base modifications include, but are not limited to, inosine, purine, pyridin-4-one, pyridin-2- one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g. 6-methyluridine), or propyne modifications. Examples of nucleic acid sugar modifications include, but are not limited to, 2'-sugar modification, e.g., T- O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-fluoroarabino, T-O- methoxyethyl nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl- trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, or 2'-deoxy nucleotides.

Oligonucleotides may include a label. Exemplary labels include, but are not limited to, fluorophore labels (including, e.g., quenchers or absorbers), non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass-modifying groups, affinity labels, magnetic particles, antigens, enzymes

(including, e.g., peroxidase, phosphatase), substrates, and the like. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Affinity labels provide for a specific interaction with another molecule. Examples of affinity labels include, for instance, biotin, avidin, streptavidin, dinitrophenyl, digoxigenin, cholesterol, polyethyleneoxy, haptens, and peptides. In some embodiments, the label may be incorporated into the oligonucleotide by using labeled deoxyribonucleotide triphosphates (dNTP's) when synthesizing the oligonucleotides. In certain aspects a label is a fluorophore. Fluorophore labels include, but are not limited to, dyes of the fluorescein family, the carboxyrhodamine family, the cyanine family, and the rhodamine family. Other families of dyes that can be used in the invention include, e.g., polyhalofluorescein-family dyes, hexachlorofluorescein- family dyes, coumarin-family dyes, oxazine-family dyes, thiazine-family dyes, squaraine-family dyes, chelated lanthanide-family dyes, the family of dyes available under the trade designation ALEXA FLUOR from Molecular Probes, and the family of dyes available under the trade designation BODIPY, from Invitrogen (Carlsbad, CA). Dyes of the fluorescein family include, e.g., 6-carboxyfluorescein (FAM), 2',4',1,4,- tetrachlorofluorescein (TET), 2^>,4^>,5',7^>,l,4-hexachlorofluorescein (HEX), T, T- dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE), 2'-chloro-5'-fluoro-7',8'-fused phenyl- 1 ,4-dichloro-6-carboxyfluorescein (NED), 2'-chloro-7'-phenyl- 1 ,4-dichloro-6- carboxyfluorescein (VIC), 6-carboxy-X-rhodamine (ROX), and 2',4',5',7'-tetrachloro-5- carboxy- fluorescein (ZOE). Dyes of the carboxyrhodamine family include tetramethyl- 6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), Texas Red,

Rl 10, and R6G. Dyes of the cyanine family include Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7. Fluorophores are readily available commercially from, for instance, Perkin-Elmer (Foster City, Calif), Molecular Probes, Inc. (Eugene, Oreg.), and Amersham GE Healthcare (Piscataway, N.J.). The label may be a quencher. Quenchers may be fluorescent quenchers or non- fluorescent quenchers. Fluorescent quenchers include, but are not limited to, TAMRA, ROX, DABCYL, DABSYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole,and nitroimidazole compounds. Exemplary non-fluorescent quenchers that dissipate energy absorbed from a fluorophore include those available under the trade designation BLACK HOLE, from

Biosearch Technologies, Inc. (Novato, CA), those available under the trade designation ECLIPSE DARK, from Epoch Biosciences (Bothell, WA), those available under the trade designation QXL, from Anaspec, Inc. (San Jose, CA), and those available under the trade designation IOWA BLACK, from Integrated DNA Technologies (Coralville, Iowa).

Typically, a fluorophore and a quencher are used together, and may be on the same or different oligonucleotides. When paired together, a fluorophore and fluorescent quencher can be referred to as a donor fluorophore and acceptor fluorophore, respectively. A number of convenient fluorophore/quencher pairs are known in the art (see, for example, Glazer et al, Current Opinion in Biotechnology, 1997;8:94-102; Tyagi et al., Nat. BiotechnoL, 1998;16:49- 53) and are readily available commercially from, for instance, Molecular Probes (Junction City, OR), and Applied Biosystems (Foster City, CA). Examples of donor fluorophores that can be used with various acceptor fluorophores include, but are not limited to, fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido- 4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1-pyrenebutyrate, and 4- acetamido-4'-isothiocyanatostilbene-2- ,2'-disulfonic acid derivatives. Acceptor fluorophores typically depend upon the donor fluorophore used. Examples of acceptor fluorophores include, but are not limited to, LC-Red 640, LC-Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorophores are readily available commercially from, for instance, Molecular Probes or Sigma Chemical Co. (St. Louis, MO).

Examples of probes useful in real-time assays using donor and acceptor fluorophores include, but are not limited to, adjacent probes (Cardullo et al., Proc. Natl. Acad. Sci. USA, 1988;85:8790- 8794; Wittwer BioTechniques, 1997;22:130- 131), and Taqman probes (Holland et al., Proc. Natl. Acad. Sci. USA, 1991;88:7276- 7280; Livak et al., PCR Methods Appl 1995;4:357-62). Examples of probes and primers useful in real-time assays using fluorphores and non- fluorescent quenchers include, but are not limited to, molecular beacons (Tyagi et al., Nat. BiotechnoL, 1996;14:303-308; Johansson et al., J. Am. Chem. Soc, 2002; 124:6950-6956), scorpion primers

(including duplex scorpion primers) (Whitcombe et al., U.S. Patent 6,326,145; Whitcombe et al., Nat. BiotechnoL, 1999;17:804-817), amplifluor primers (Nazarenko et al., Proc. Natl. Acad. Sci. USA, 1997;25:2516-2521), and light-up probes (Svanvik et al., Anal. Biochem., 2000;287:179-182). Polynucleotides of the present invention may be used as probes in a real-time assay wherein one polynucleotide is labelled with a fluorophore, the other polynucleotide is labelled with a fluorescent quencher, and, when both are hybridized to a target polynucleotide, the fluorophore and fluorescent quencher are separated by about 1-5 nucleotide bases. A polynucleotide of the present invention can be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al,

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors and viral vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. Typically, a vector is capable of replication in a bacterial host, for instance E. coli. Preferably the vector is a plasmid. Vectors may also include a mms coding region, such as SEQ ID NO:3, or a portion thereof, preferably nucleotides from about 186 to about 235 of SEQ ID NO:3. Such vectors can be used as, for instance, control target polynucleotides.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryotic cells. Suitable prokaryotic cells include eubacteria, such as gram-negative microbes, for example, E. coli. Vectors can be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral- mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells. Polynucleotides of the present invention can be produced in vitro or in vivo.

For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known. Methods for in vitro synthesis also include, for instance, in vitro transcription using a circular or linear expression vector in a cell free system. Expression vectors can also be used to produce a polynucleotide of the present invention in a cell, and the polynucleotide then isolated from the cell. Methods of use

The present invention includes methods for detecting polynucleotides that are characteristic of E. sakazakii. If the sample is obtained from a subject, the method may be used to determine whether the subject is infected with E. sakazakii. The methods of this aspect of the present invention typically include contacting a target polynucleotide with a primer pair of the present invention, amplifying the polynucleotide, and detecting the resulting amplified product.

The target polynucleotide used in the methods may be present in a sample. The sample can be a food sample, a beverage sample, a fermentation broth, a forensic sample, an agricultural sample (e.g., from a plant or animal), or an environmental sample (e.g., soil, dirt, garbage, sewage, or water). Preferably, the sample is a biological sample. A "biological sample" refers to a sample obtained from eukaryotic or prokaryotic sources. Examples of eukaryotic sources include mammals, such as a human or a member of the family Muridae (a murine animal such as rat or mouse). Examples of prokaryotic sources include E. sakazakii or other microbes containing an endogenous or recombinant mms polynucleotide sequence.

The biological sample can be, for instance, in the form of a single cell, in the form of a tissue, or in the form of a fluid. Cells or tissue can be derived from in vitro culture. When obtained from an animal, the biological sample can be obtained from, for instance, anal swabs, perirectal swabs, stool samples, blood, and/or body fluids. In some aspects, the biological sample is obtained from a subject suspected of having an Enterobacter infection. A sample may be an isolated polynucleotide, for instance, a polynucleotide present in a vector as described herein, or an polynucleotide isolated using methods described hereinbelow. The sample can be a colony of microorganisms, such as a colony obtained from the surface of an agar plate or a colony obtained from a culture device sold by 3M Company (St. Paul, MN, USA) under the trade name PETRIFILM. The colony may be an axenic culture, or it may be a mixed colony of microorganisms. The sample can be obtained from a broth culture of microorganisms, which may be a monoculture or a mixed culture, as in an enrichment culture.

The sample can be a solid sample (e.g., solid tissue) that is dissolved or dispersed in water or an organic medium, or from which the polynucleotide has been extracted into water or an organic medium. For example, the sample can be an organ homogenate. Thus, the sample can include previously extracted polynucleotides. In some aspects, the sample may be incubated with an enrichment broth to enrich for E. sakazakii microorganisms that are present. The sensitivity of detection of such a microbe in a sample can be enhanced by including the enrichment culture process prior to sample preparation to extract the polynucleotides for amplification and detection. Sample material (e.g., a biological sample) is used to inoculate a suitable medium/broth. The broth can provide simple nutrient requirements to promote the growth of the target organism, or may be used to provide selective enrichment, using media supplemented with selective agents, such as antibiotics, at a certain concentration which kills other microbes in the sample but allows for proliferation of E sakazakii, and then the culture is incubated at a suitable temperature (e.g., 37°C) for a period of time (e.g., between 4 and 48 hours; preferably, between 4-24 hours). At the end of the enrichment culture process, the sample with the microbe of interest is collected from a portion of the culture by centrifugation, filtration, sampling by pipette, or other suitable methods, and then used in methods of the present invention involving amplification and detection.

The polynucleotides may be from an impure, partially pure, or a pure sample. The purity of the original sample is not critical, as polynucleotides may be obtained from even grossly impure samples. For example, polynucleotides may be obtained from an impure sample of a biological fluid such as blood, saliva, feces, or tissue. If a sample of higher purity is desired, the sample may be treated according to any conventional means known to those of skill in the art prior to undergoing the methods of the present invention. A polynucleotide may be isolated using methods described hereinbelow.

Complex biological samples (feces, blood, food, tissue, sputum, etc.) may contain solid debris and/or amplification inhibitors. Solid debris is commonly removed by sedimentation or centrifugation (separate supernatant from solids), filtration, etc. Amplification inhibitors are often removed by treatment with protein denaturants or proteases, dilution, etc. Undesired polynucleotide-containing cells may be reduced by selective lysis, differential centrifugation, filtration, etc.

Specific microbes, preferably, species from the Enterobacteriaceae family or Enterobacter genus, may be concentrated or enriched from a sample prior to amplifϊcation of a target polynucleotide to detect the presence of E. sakazakii. For example, a biological sample can be exposed to a matrix functionalized with an agent that will interact with E. sakazakii, but not interact with other components present in a biological sample. The interaction is a reversible retention via a wide variety of mechanisms, including weak forces such as Van der Waals interactions, electrostatic interactions, affinity binding, or physical trapping. Examples of useful agents include, but are not limited to, specific interactions, such as those mediated by an anti- Enterobacteriaceae antibody, and non-specific interactions. Examples of agents that can be used to mediate non-specific interactions with the Enterobacteriaceae family include silica, zirconia, alumina beads, metal colloids such as gold, and gold coated sheets that have been functionalized through mercapto chemistry, for example (Parthasarathy, U.S. Provisional Application Serial Number 60/913,813, filed April 25, 2007, Attorney Docket No. 62470US002).

Agents that interact with E. sakazakii can be present on any solid phase material. Examples include polyolefin, polystyrene, nylon, poly(meth)acrylate, polyacrylamide, polysaccharide, and fluorinated polymers, as well as resins such as agarose, latex, cellulose, and dextran. The solid material may be in any form, preferably in the form of particulate material (e.g., particles, beads, microbeads, microspheres) or any other form (e.g., fibrils) that can be introduced into a micro fluidic device (Parthasarathy, U.S. Provisional Application Serial Number 60/913,813, filed

April 25, 2007, Attorney Docket No. 62470US002).

Polynucleotides present in a sample may be introduced directly into the amplification reaction. Prior to use in an amplification reaction, polynucleotides present in a sample, such as a biological sample, may be prepared for amplification. Treatments for preparing polynucleotides for amplification are well known in the art and used routinely. Polynucleotides can be extracted from a biological sample. Extraction typically includes lysis of microorganisms to release polynucleotides. Lysis herein is the physical disruption of the membranes of the cells. Extraction can be accomplished by the use of standard techniques and reagents. Examples include, for instance, heat treatment, boiling, hydrolysis with proteinases, exposure to ultrasonic waves, detergents, strong bases, or organic solvents such as phenol chloroform (Lin et al, U.S. Pat. No. 5,620,852; Kellogg et al, U.S. Patent No. 5,010,183). Polynucleotides can be prepared by use of particles, such as magnetic glass particles, under conditions to bind the polynucleotides, followed by washing to remove impurities, and then obtaining purified polynucleotides with a wash designed to remove the bound polynucleotides (MagNA Pure, International Publication No. WO 01/37291 Al).

The polynucleotides used as targets in the methods of the present invention may be of any molecular weight and in single-stranded form, double-stranded form, circular, plasmid, etc. Various types of polynucleotides can be separated from each other (e.g., RNA from DNA, or double-stranded DNA from single-stranded DNA). For example, polynucleotides of at least about 100 bases in length, longer molecules of 1000 bases to 10,000 bases in length, and even high molecular weight nucleic acids of up to about 3.2 megabases can be used in the methods of the present invention.

Polynucleotide amplification, such as the polymerase chain reaction (PCR), is a method for the enzymatic amplification of specific segments of polynucleotides. The amplification is based on repeated cycles of the following basic steps: denaturation of double-stranded polynucleotides, followed by primer annealing to the target polynucleotide, and primer extension by a polymerase (Mullis et al., U.S. Patent

4,683,195, Mullis, U.S. Patent 4,683,202, and Mullis et al., U.S. Patent 4,800,159). The primers are designed to anneal to opposite strands of the DNA, and are positioned so that the polymerase-catalyzed extension product of one primer can serve as the template strand for the other primer. The amplification process can result in the exponential increase of discrete polynucleotide fragments whose length is defined by the 5' ends of the primer pairs.

Generally, these steps are achieved in a cycling step. A typical cycling step used in DNA amplification involves two target temperatures to result in denaturation, annealing, and extension. The first temperature is an increase to a predetermined target denaturation temperature high enough to separate the double-stranded target polynucleotide into single strands. Generally, the target denaturation temperature of a cycling step is approximately 92°C to 98°C, such as 94°C to 96°C, and the reaction is held at this temperature for a time period ranging between 0 seconds to 5 minutes. The temperature of the reaction mixture is then lowered to a second target temperature. This second target temperature allows the primers (and probe(s), if present) to anneal or hybridize to the single strands of DNA, and promote the synthesis of extension products by a DNA polymerase. Generally, the second temperature of a cycling step is approximately 57°C to 63°C, such as 59°C to 6PC, and the reaction is held at this temperature for a time period ranging between 0 seconds to 1 minute. This second temperature and time can vary greatly depending upon the primers (and probe(s), if present) and target polynucleotide used. This completes one cycling step. The next cycle then starts by raising the temperature of the reaction mixture to the denaturation temperature. Typically, the cycle is repeated to provide the desired result, which may be to produce a quantity of DNA and/or detect an amplified product. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of polynucleotides, more cycling steps may be required to amplify the target polynucleotide sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40,

60, or even 100 times. In some instances (e.g., real time PCR), each cycle comprises detecting the product. Examples of detection methods are known in the art and some are discussed in more detail below. As will be understood by the skilled artisan, the above description of the thermal cycling reaction is provided for illustration only, and accordingly, the temperatures, times and cycle number can vary depending upon the nature of the thermal cycling reaction and application.

Optionally, a third temperature is also used in a cycling step. The use of three target temperatures also results in denaturation, annealing, and extension, but separate target temperatures are used for the denaturation, annealing, and extension. When three target temperatures are used the annealing temperatures generally range from 45°C to

60⁰C, depending upon the application. The third target temperature is for extension, is typically held for a time period ranging between 30 seconds to 10 minutes, and occurs at a temperature range between the annealing and denaturing temperatures (e.g., generally between 68 and 72 degrees C). A person skilled in the art will recognize that several factors, such as the primer annealing temperature, salt concentration, the overall complementarity between the primers and the target polynucleotide, and, in particular, the complementarity between the nucleotides proximal the 3' end of the primer and the corresponding nucleotides in the target polynucleotide.

DNA polymerases for use in the methods and compositions of the present invention are capable of effecting extension of a primer according to the methods of the present invention. Accordingly, a preferred polymerase is one that is capable of extending a primer along a target polynucleotide. Preferably, a polymerase is thermostable. A thermostable polymerase is a polymerase that is heat stable, i.e., the polymerase catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Useful thermostable polymerases are well known and used routinely. Thermostable polymerases have been isolated from Thermusflavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus .

A polymerase typically initiates synthesis at the 3 '-end of a primer annealed to a target polynucleotide, and proceeds in the 5'-direction along the target polynucleotide. A polymerase may possess a 5' to 3' exonuc lease activity, and hydro lyze intervening, annealed probe(s), if present, to release portions of the probe(s), until synthesis terminates. Examples of suitable polymerases having a 5' to 3' exonuclease activity include, for example, Tfϊ, Taq, and FastStart Taq (Roche). In other aspects, the polymerase has little or no 5' to 3' exonuclease activity so as to minimize degradation of primer, termination or primer extension polynucleotides. This exonuclease activity may be dependent on factors such as pH, salt concentration, whether the target is double stranded or single stranded, and so forth, all of which are familiar to one skilled in the art. Examples of suitable polymerases having little or no 5' to 3' exonuclease activity include Klentaq (Sigma, St. Louis, MO). Typically, amplification involves mixing one or more target polynucleotides which can have different sequences with a "master mix" containing the reaction components for performing the amplification reaction and subjecting this reaction mixture to temperature conditions that allow for the amplification of the target polynucleotide. The reaction components in the master mix can include a buffer which regulates the pH of the reaction mixture, magnesium ion, one or more of the natural nucleotides (corresponding to adenine, cytosine, guanine, and thymine or uracil, often present in equal concentrations), that provide the energy and nucleosides necessary for the synthesis of an amplification product, primer pairs that bind to the target in order to facilitate the initiation of polynucleotide synthesis, a polymerase that adds the nucleotides to the complementary strand being synthesized, and optionally, one or more probes. One skilled in the art will recognize that a successful amplification reaction will not occur in the absence of a target polynucleotide, although the presence of a target polynucleotide is not required to perform the present methods. The presence or absence of an amplified product can be determined or its amount measured. Detecting an amplified product can be conducted by standard methods well known in the art and used routinely. The detecting may occur, for instance, after multiple amplification cycles have been run, or during each amplification cycle (typically referred to as real-time). Detecting an amplification product after multiple amplification cycles have been run is easily accomplished by, for instance, resolving the amplification product on a gel and determining whether the expected amplification product is present. In order to facilitate real-time detection or quantification of the amplification products, one or more of the primers used in the amplification reaction can be labeled, and various formats are available for generating a detectable signal that indicates an amplification product is present. The most convenient label is typically fluorescent, which may be used in various formats including, but are not limited to, the use of donor fluorophore labels, acceptor fluorophore labels, fluorophores, quenchers, and combinations thereof. The types of assays using the various formats may include the use of one or more primers that are labeled (for instance, scorpions primers, amplifluor primers). The skilled person will understand that in addition to these known formats, new types of formats are routinely disclosed. The present invention is not limited by the type of method or the types of primers used to detect an amplified product. Using appropriate labels (for example, different fluorophores) it is possible to combine (multiplex) the results of several different primer pairs in a single reaction.

As an alternative to detection using a labeled primer, an amplification product can be detected using a polynucleotide binding dye such as a fluorescent DNA binding dye. Examples include, for instance, SYBRGreen or SYBRGoId (Molecular Probes). Upon interaction with the double-stranded amplification product, such polynucleotide binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A polynucleotide binding dye such as a polynucleotide intercalating dye also can be used.

The present invention may be coupled with detection systems and methods involving separation and detection of the amplification products, such as the methods and detection system components described in PCT/IB2007/000923, filed on April 10, 2007, entitled NUCLEIC ACID DETECTION USING LATERAL FLOW METHODS, and in PCT Publication No. WO 2008/032205. As described therein, the first and second primer sequences are labelled with first and second labels, respectively. Preferably, the first and second labels are selected from haptens such as, for example, biotin, fluorescein derivatives (e.g. FITC), rhodamine derivatives (e.g. TAMRA), Cascade Blue, Lucifer yellow, 5-bromo-2-deoxyuridine (BrdU), dinitrophenol (DNP), digoxygenin (DIG), and short peptide label sequences (i.e. short peptides against which specific antibodies can be raised). In some embodiments, the first label may be biotin and the second label may be DNP, in which case, amplicons generated during the amplification step are labelled with both biotin and DNP. In other embodiments, the primers are labelled with labelled deoxyribonucleotide triphosphates (dNTPs) such as, for example, labelled 2'-deoxyadenosine 5'-triphosphate (dATPs) and/or labelled T- deoxythymidine triphosphate (dTTPs). During the subsequent separation step, the amplicons can be captured on solid surfaces (e.g. chromatographic substrates, such as membranes, or microparticles) by, for example, antibodies, antibody fragments, receptors, or other binding partners, and detected using a system appropriate for the labels that are incorporated in the primers. Detection system components include devices for capturing and detecting the amplicons, such as lateral flow devices and flow-through devices.

Microparticles are preferably composed of one or more substantially inert substances such as gold, silica, selenium, polystyrene, melamine resin, polymethacrylate, styrene/divinylbenzene copolymer, and polyvinyltoluene. The microparticles are preferably non-porous. The microparticles may comprise a substance to allow for visualisation of results at the test and control regions of the substrate. Conveniently, such a substance will be a dye or other colored substance to allow for visualisation with the unaided eye, however alternatively, the substance may be, for example, a label substance allowing visualisation through the generation of a coloured substance (eg an enzyme or other catalytic-label) or by fluorescence, luminescence or magnetic interactions (e.g. using a fluorimeter, luminometer or magnetic induction). The microparticles may be of a diameter size in the range of 0.002 to 5 μm. Preferably, the microparticles are gold microparticles having a diameter size in the range of 0.002 to 0.25 μm (i.e., 2 to 250 nm), more preferably 0.01 to 0.06 μm (ie 10 to 60 nm), and most preferably having an average diameter size of 0.04 μm (ie 40 nm). Suitable polystyrene microparticles include those having a diameter size in the range of 0.1 to 5 μm. Controls can be included when an amplification reaction is run. Control target polynucleotides can be amplified from a positive control sample (e.g., a target polynucleotide other than mms) using, for example, control primers. Positive control samples can also be used to amplify a target mms polynucleotide. Such a control can be amplified internally (e.g., within each amplification reaction) or in separate samples run side -by-side with a subject's sample. Each run may also include a negative control that, for example, lacks a target mms.

It is understood that the present invention is not limited by the device used to conduct the amplification and detection of the amplified product. For example, suitable devices may include conventional amplification devices such as, for instance, the

LIGHTCYCLER (Roche) (University of Utah Research Foundation, International Publication Nos. WO 97/46707, WO 97/46714, and WO 97/46712), MX3005p (Stratagene, La Jolla, CA), and amplification devices available from Bio-Rad. It may be preferred that the present invention is practiced in connection with a microfluidic device. "Microfluidic" refers to a device with one or more fluid passages, chambers, or conduits that have at least one internal cross-sectional dimension, e.g., depth, width, length, diameter, etc., that is less than 500 μm, and typically between 0.1 μm and 500 μm. Typically, a microfluidic device includes a plurality of chambers (e.g., amplification reaction chambers, loading chambers, and the like), each of the chambers defining a volume for containing a sample. Some examples of potentially suitable microfluidic devices are described in U.S. Publication Numbers 2002/0064885 (Bedingham et al); US2002/0048533 (Bedingham et al); US2002/0047003 (Bedingham et al.); and US2003/138779 (Parthasarathy et al.); as well as U.S. Patent Nos. 6,627,159 (Bedingham et al.); 6,720,187 (Bedingham et al.); 6,734,401 (Bedingham et al.); 6,814,935 (Harms et al.); 6,987,253 (Bedingham et al.); 7,026,168

(Bedingham et al.); and 7,164,107 (Bedingham et al.). The present invention also includes methods for isolating, preferably, purifying a polynucleotide. The methods of this aspect of the present invention typically include providing a mixture that contains single stranded polynucleotides, exposing the mixture to an oligonucleotide of the present invention under suitable conditions for specific hybridization of the oligonucleotide to a single stranded polynucleotide to result in a hybrid, and isolating the hybrid from non- hybridized single stranded polynucleotides. Such methods may be used to prepare a sample prior to amplification of a target polynucleotide present in E. sakazakii. In certain embodiments, after exposing the oligonucleotide under suitable conditions to form a hybrid, the method further comprises attaching the oligonucleotide to a solid phase material, such as a membrane or a microparticle.

The mixture may be obtained from a sample, preferably, a biological sample. Typically, the sample may contain E. sakazakii. The sample may be any suitable sample including, for example, a food sample, a sample prepared from a swab of a food preparation surface, a waste or process water sample, and a micro-organism culture or enrichment sample (e.g. a sample aliquot from the first or third tubes of a test or system sold by 3M Tecra under the tradenames UNIQUE, or IMMUNOCAPTURE, from a colony obtained from the surface of an agar plate or culture device sold by 3M

Company (St. Paul, MN, USA) under the trade name PETRIFILM). The sample may be prepared for isolation by extraction as described hereinabove. The polynucleotides in the mixture may be impure (e.g., other cellular materials and/or solid debris are present), partially pure, or purified. The polynucleotides in the mixture may be denatured using well known and routine methods. Examples of such methods include, for instance, heating, or exposure to alkaline conditions.

The mixture of single stranded polynucleotides is exposed to an oligonucleotide of the present invention in suitable conditions for specific hybridization of the oligonucleotide and the complementary single stranded polynucleotide. The oligonucleotide typically includes a label, preferably an affinity label. Conventional hybridization formats which are particularly useful include those where oligonucleotide is immobilized on a solid support (solid-phase hybridization) and those where the polynucleotides, (both single stranded polynucleotides and oligonucleotides) are all in solution (solution hybridization). In solid-phase hybridization formats, the oligonucleotide is typically attached to a solid phase material prior to the hybridization. In solution hybridization formats, the oligonucleotide is typically attached to a solid phase material after the hybridization. In both formats, the attachment is mediated by a label, preferably an affinity label, that is attached to the oligonucleotide. Examples of useful solid phase materials include, for instance, polyolefm, polystyrene, nylon, poly(meth)acrylate, polyacrylamide, polysaccharide, and fluorinated polymers, as well as resins such as agarose, latex, cellulose, and dextran. The solid material may be in any form, preferably in the form of particulate material (e.g., particles, beads, microbeads, microspheres) or any other form (e.g., fibrils) that can be introduced into a microfluidic device (Parthasarathy, U.S. Provisional Application Serial Number 60/913,813, filed April 25, 2007, Attorney Docket No. 62470US002).

The hybridization is performed under suitable conditions for selectively binding the labeled oligonucleotide to the substantially complementary, preferably complementary, single stranded polynucleotides present in the mixture, e.g., stringent hybridization conditions. General methods for hybridization reactions and probe synthesis are disclosed in Molecular Cloning by T. Maniatis, E. F. Fritsch and J. Sambrook, Cold Spring Harbor Laboratory, 1982, for example the hybridization conditions include the use of a hybridization buffer such as 6x SSC, 5x Denhardt's reagent, 0.5% [w/v] SDS, and a blocking reagent such as 100 μg/ml salmon sperm. Hybridization may be allowed to occur at 68°C for at least 2 hours. After the hybridization, (and attachment of the labeled oligonucleotide, if appropriate), the non- hybridized polynucleotides, and any other materials that may be present, can be removed by washing at room temperature several times in a solution containing 2x SSC and 0.5% SDS. Optionally, the isolated polynucleotide may be purified by denaturing the hybrid to release the isolated polypeptide and removing the bound oligonucleotide and solid support.

Kits

The present invention provides kits, which can include oligonucleotides of the present invention, such as, for instance, a primer pair, and optionally, a probe. Other components that can be included within kits of the present invention include conventional reagents such as a master mix; hybridization solutions; detection system components such as solid phase supports (e.g., a membrane, a microparticle), flow- through devices, lateral flow devices,and external positive or negative controls; and the like.

The kits typically include packaging material, which refers to one or more physical structures used to house the contents of the kit. The packaging material can be constructed by well-known methods, preferably to provide a contaminant-free environment. The packaging material may have a marking that indicates the contents of the kit. In addition, the kit contains instructions indicating how the materials within the kit are employed. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like.

"Instructions" typically include a tangible expression describing the various methods of the present invention, including sample preparation conditions, amplification conditions, and the like.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

Detection of Enterobacter sakazakii in Pure Culture Samples Using Gene-Specific Primers

Materials and Methods

Sample: A pure culture of E. sakazakii (food isolate, Tecra International Culture

Collection #4217) was grown overnight in buffered peptone water and was heat-killed by boiling the culture for 15 minutes. A 1 μl sterile loop was used to transfer sample and inoculate the PCR mix.

PCR primers

Primers were selected to enable PCR amplification of a region of E. sakazakii. The primers selectively amplify a 50 bp region of the macromolecular synthesis (MMS) operon (see Genbank accession number LO 1755 for partial sequence). The nucleotide sequences of the primers that were used in this experiment are shown in Table 1.

Table 1 : Primer and target DNA sequences for the detection of the mms gene in E. sakazakii.. SEQ ID NO:1 and SEQ ID NO:2 correspond to the forward and reverse primers, respectively, in the PCR amplification reaction. SEQ ID NO:3 corresponds to the nucleotide sequence disclosed in Genbank accession number L01755 .

Name Sequence 5'-3'

1 GTACT AATTCCTCAG GGGATATT

SEQ ID NO: 1 (corresponds to nucleotides 186-208 in SEQ ID NO:3) 1 ACTACTACTC TGTCTGTTTC AGGGG (corresponds to the complement of nucleotides 211-235 in

SEQ ID NO:2 SEQ ID NO:2)

1 AACGAGCCGT TCGACGTAGC ACTGCGTCGC

31 TTCAAACGTT CCTGCGAGAA AGCGGGTGTT 61 CTGGCGGAAG TTCGTCGTCG TGAATTCTAT 91 GAAAAACCGA CTACCGAACG TAAACGCGCC 121 AAAGCTTCCG CAGTGAAACG TCACGCGAAG 151 AAACTGGCTC GCGAAAACGC ACGCCGTACT

SEQ ID NO:3 181 CGTCTGT ACT AATTCCTCAG GGGATATTGT 211 CCCCTGAAAC AGACAGAGTA GTAGTTGTAG 241 AGGCCGTGCT TCCGAAAGGA ATGCGCGGCT 271 TATTCTCGTT TATGGGCTGA TAAAAACGGG 301 GCTTATGGCT GGACGAATC

The forward primer (SEQ ID NO: 1) was labelled at the 5' end with biotin and the reverse primer (SEQ ID NO:2) was labelled at the 5' end with fluorescein. The labelled primers were obtained from Gene Works (Hindmarsh SA, Australia).

Flow through device Flow-through devices were constructed as described in PCT Publication No.

WO 2008/032205. The detection membrane was prepared using nitrocellulose membrane (BA-83, Whatman). Test anti-FITC antibody (catalog number F5636, Sigma, MO, USA) was diluted to 0.3 mg/ml in Striping Solution (Millenia Dignostics, CA, USA)] and approximately one microliter (per test unit) was applied in stripe format across the nitrocellulose membrane using a Bio Jet Quanti (BioDot, Irvine CA, USA) dispenser. The membrane was dried at room temperature, blocked for one hour at room temperture using Lateral flow Blocking Buffer (Millenia Diagnostics, CA, USA), dried at room temperature and stored until use. Prior to use, the membranes were cut to size (approximately 1.2 cm wide and 1.8 cm long) and used to assemble a detection cassette containing an absorbent pad (Pall-197, Pall, NY, USA) with a 0.8 cm diameter aperture as described in PCT Publication No. WO 2008/032205. The side containing striped antibody faced the aperture of the cassette.

Amplification

PCR amplification was conducted as follows using the primers described above (ie having the nucleotide sequences of SEQ ID NO: 1 and 2):

(i) Dried PCR mix (e.g. Bioneer Accupower, Korea) was rehydrated using 20 microliters of sterile, molecular quality H2O containing the primers (0.5 μM of each primer, SEQ ID NO: 1 and 2).

(ii) 1 μl of sample was inoculated into the rehydrated PCR mix using a sterile 1 μl loop.

(iii) Using a Mastercycler Personal (Eppendorf, Germany) thermocycler the inoculated PCR mix was subjected to an initial heating step of 94⁰C for 4 minutes; followed by

(iv) Subjecting the inoculated PCR mix to 40 cycles of a. melting step, 94 ° C for 20 seconds, b. annealing step, 59⁰ C for 20 seconds, and c. elongation step, 72⁰ C for 20 seconds, (v) A final elongation step at 72 ° C for 1 minute (vi) Held at 8 ° C until flow-through assay step

The entire PCR amplification reaction took under 90 minutes.

Preparation of PCR product

Goat anti-biotin antibody-coated gold microparticles were obtained from Alchemy Laboratories, Dundee, U.K. Just prior to application to the flow-through device, a 5 μl aliquot of the PCR product was mixed with 100 μl running buffer comprising phosphate buffered saline (PBS, pH 7.4) and Tween-20 (0.05%). Seven microliters of the gold microparticles (OD530 =10.2) were added to the diluted PCR product and the labeled PCR products were allowed to adsorb to the microparticles.

Assaying on the flow through device First, approximately 100 μl of PBS-0.05% Tween 20 was applied to pre-wet the membrane. After the pre -wetting solution flowed through the membrane, the buffered PCR product sample was applied to the membrane within the device and allowed to pass through the membrane to the absorbent pad. Finally, a wash step involving applying approximately 100 μl of PBS-0.05% Tween 20 was carried out. The results were visually interpreted.

Results and Discussion

A clear pink colored stripe was evident on the membrane indicating the presence of doubly-labeled PCR product (i.e., labeled with both biotin and FITC label), and a positive result. A white background indicated that the membrane was washed sufficiently to remove unbound gold microparticles. From the initial heating of the sample to the appearance of the results on the membrane took approximately 100 minutes.

Example 2

Specific detection of several Enterobacter sakazakii and exclusion of non-ii. sakazakii strains using labeled primer sequences directed to the mms operon

Materials and Methods

The bacterial strains used in this example are listed in Table 2. E. sakazakii and non-ii. sakazakii strains were grown overnight in TSB at 37⁰C. Glycerol was added to each cell suspension to a final concentration of 25% (v/v) and the suspensions were stored frozen at -8O⁰C until they were used in the experiments.

Table 2. Strain List. All strain numbers are Tecra International Culture Collection (TICC) strain numbers, unless noted otherwise. Strains in the TICC were isolated from various sources, including food isolates and isolates donated /obtained from third parties. ATCC is a registered trademark of the American Type Culture Collection (Manassas, VA, USA).

PCR primers Polymerase chain reaction (PCR) primers were prepared as described in

Example 1. The labeled and desalted primers were obtained from Geneworks (Hindmarsh SA, Australia). The forward primer (SEQ ID NO: 1) was labeled at the 5' end with biotin while the reverse primer (SEQ ID NO:2) was labeled at the 5' end with FITC. PCR amplification with these primers produced an amplicon of 50 nucleotides in length.

Lateral flow device

A lateral flow device was prepared using a strip of nitrocellulose membrane (Immunopore FP, Whatman) of approximately 5 mm x 60 mm in dimensions. A sample pad (Arista Biologicals, Allentown, PA, USA) was applied to the strip to allow loading of the buffered assay sample. At the distal end of the device, an absorbent pad comprising cotton fibre (Arista Biochemicals, Allentown, PA, USA) was adhered to draw the flow of the buffered assay sample across the membrane. The test line was prepared by adsorbing 0.46 μg anti-FITC monoclonal antibodies (Sigma-Aldrich, St. Louis, MO, USA) to the membrane in a thin line across the width of the membrane. The entire lateral flow device was constructed by applying the membrane and sample and absorbent pads onto an adhesive backing card (Millenia Diagnostics, San Diego, CA, USA). Amplifϊcation

PCR amplification was conducted in accordance with methods well known to persons skilled in the art, as described below. With the primers described above (i.e. SEQ ID NO: 1 and 2), the PCR amplification was conducted as follows: (i) Dried PCR mix (Accupower, Bioneer, Korea) was rehydrated using

20 μl molecular quality H₂O comprising a final concentration of 0.5 μM of each primer (SEQ ID NOS: 1 and 2)

(ii) A 1 μl loop of sample was inoculated using a sterile loop into the rehydrated dried PCR mix; (iii) Using a, Eppendorf MasterCycle Personal thermal cycler, the inoculated PCR mix was subjected to an initial heating step of 94⁰C for 4 minutes; followed by

(iv) 35 cycles of a. melting step, 94 ° C for 15 seconds, b. annealing step, 6O ⁰ C for 20 seconds, and c. elongation step, 72⁰ C for 20 seconds.

Following PCR, samples were held briefly at 8⁰C until use.

Preparation of PCR product Goat anti-biotin antibody-coated gold microparticles were obtained from

Alchemy Laboratories, Dundee, U.K. Just prior to application to the flow-through device, a 5 μl aliquot of the PCR product was mixed with 100 μl running buffer comprising phosphate buffered saline (PBS, pH 7.4) and Tween-20 (0.05%). Seven microliters of the gold microparticles (OD530 =10.2) were added to the diluted PCR product and the labeled PCR products were allowed to adsorb to the microparticles.

Assaying on the lateral flow device

The buffered assay sample, comprising the entire 115 μl aliquot of the buffered PCR product/gold microparticle mixture, was loaded onto the sample pad of the lateral flow device as described above. The constituents of the mixture were allowed to flow across the membrane for 5 minutes. The test line comprising anti-FITC antibodies "trapped" doubly labeled amplicons present in the mixture that were labeled with FITC. Doubly labeled amplicons were bound to gold microparticles, and thus, when trapped at the test line by anti-FITC antibodies, generated a pinkish-red line.

Results and Discussion

All E. sakazakii strains were readily detected by the presence of a pinkish-red line using the labeled primers and lateral flow detection method. Strains that should have been excluded from detection (i.e, all strains unrelated to E. sakazakii) did not display a pinkish-red line in the test region thereby indicating that the primers were specific for E. sakazakii and did not amplify the selected non-E. sakazakii strains. One strain, known to display some characteristics of E. sakazakii (TICC # 2798, Enterobacter sp. amnigenus cloacae sakazakii) was detected using these primers indicating that this organism has a mms operon sequence complementary to the described primers .

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in

GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A method for detecting E. sakazakii in a biological sample, comprising: amplifying a target polynucleotide present in a biological sample to result in an amplified product, wherein the biological sample is contacted with a first mms primer and a second mms primer under suitable conditions to result in an amplified product, wherein the first primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, and the second primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3; and detecting the amplified product, wherein the presence of the amplified product is indicative of the presence of E. sakazakii in the biological sample.

2. A method for detecting the absence of E. sakazakii in a biological sample, comprising: amplifying a target polynucleotide present in a biological sample to result in an amplified product, wherein the biological sample is contacted with a first mms primer and a second mms primer under suitable conditions to result in an amplified product, wherein the first primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO: 1 , and the second primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3; and detecting the absence of the amplified product, wherein the absence of the amplified product is indicative of the absence of E. sakazakii in the biological sample

3. The method of claim 1 or 2 wherein amplifying a target polynucleotide comprises repeated cycles.

4. The method of claim 1 or 2 wherein detecting the amplified product comprises detecting the amplified product during each cycle.

5. The method of claim 1 or 2 wherein the biological sample is suspected of containing E. sakazakii.

6. The method of claim 1 or 2 wherein the biological sample is selected from the group consisting of a eukaryotic source, a prokaryotic source, a single cell, a tissue, a fluid, a food, a food ingredient, a food residue, a beverage, a beverage ingredient, a beverage residue, and water.

7. The method of claim 1 or 2 further comprising obtaining the biological sample.

8. The method of claim 1 or 2 wherein the detecting comprises detecting a fluorophore.

9. The method of claims 1 or 2 wherein the amplifying comprises a DNA polymerase comprising 5' to 3' exonuclease activity.

10. A method for isolating a polynucleotide comprising: providing a mixture comprising single stranded polynucleotides; exposing the mixture to an oligonucleotide under conditions suitable for specific hybridization of the oligonucleotide to a single stranded polynucleotide to result in a hybrid, wherein the oligonucleotide comprises a nucleotide sequence selected from at least about 80% identity to SEQ ID NO:1 or at least about 80% identity to SEQ ID NO:2, and wherein the oligonucleotide comprises an affinity label; and washing the hybrid.

11. The method of claim 10 further comprising attaching the oligonucleotide to a solid phase material after the exposing.

12. The method of claim 10 wherein the oligonucleotide is attached to a solid phase material before the exposing.

13. The method of claim 10 wherein the mixture is obtained from a biological sample.

14. A kit comprising packaging materials, a first mms primer and a second mms primer, wherein the first primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, and the second primer comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the primer pair amplifies nucleotides 186 - 235 of SEQ ID NO:3.

15. The kit of claim 14 wherein the first primer comprises SEQ ID NO:1 and the second primer comprises SEQ ID NO:2.

16. The kit of claim 14 further comprising a detection system component selected from the group consisting of a lateral flow device, a flow-through device, a membrane, and a microparticle.

17. An isolated polynucleotide wherein the polynucleotide is a product of DNA amplification and wherein the polynucleotide is about 50 base pairs in length.

18. An isolated first polynucleotide wherein the first polynucleotide comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:1, wherein the first polynucleotide amplifies a second polynucleotide comprising nucleotides 186 - 235 of SEQ ID NO:3 when used with SEQ ID NO:2.

19. An isolated first polynucleotide wherein the first polynucleotide comprises a nucleotide sequence with at least about 80% identity to SEQ ID NO:2, wherein the first polynucleotide amplifies a second polynucleotide comprising nucleotides 186 - 235 of SEQ ID NO:3 when used with SEQ ID NO: 1.

20. A solid support comprising an amplified polynucleotide with at least about 80% identity to nucleotides 186 - 235 of SEQ ID NO:3.

21. The solid support of claim 20 wherein the amplified polynucleotide is about

50 base pairs long.

22. The solid support of claim 20 or 21 wherein the solid support comprises a membrane.

23. The solid support of claim 20 or 21 wherein the solid support comprises a microparticle.