CN103930564A - Detection and quantification of lactic acid producing bacteria in food products - Google Patents
Detection and quantification of lactic acid producing bacteria in food products Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
Methods and kits for rapidly detecting and/or quantifying lactic acid bacteria using real time PCR including a first primer, a second primer, and a probe. Each of the primers and the probe are complementary to separate regions of the 16S rRNA gene of lactic acid bacteria. The methods and kits may be used for the detection and/or quantification of lactic acid bacteria in food such as yogurt, to support product labeling, and in salsa.
Description
right of priority
The application requires to enjoy the right of priority of U.S. Provisional Patent Application that the title submitted on June 27th, 2011 is " in food the detection of lactic acid producing bacteria and quantitatively " number 61/501,470, and its overall content is incorporated to herein by reference.
background
Produce lactic acid and be called milk-acid bacteria as the bacterial flora of the end product of carbohydrate metabolism.These bacteriums are found in occurring in nature, as in rotten plant, and at some food, in Yoghourt.They for example comprise and belonging to: lactobacillus (
lactobacillus), leuconos toc (
leuconostoc), Pediococcus (
pediococcus), lactococcus (
lactococcus), streptococcus (
streptococcus), Aerococcus (
aerococcus), meat Bacillaceae (
carnobacterium), enterococcus spp (
enterococcus), wine Coccus (
oenococcus), Sporolactobacillus (
sporolactobacillus), Tetracoccus (
teragenococcus), roaming Coccus (
vagococcus) and Wei Si Bordetella (
weisella) bacterium.
Milk-acid bacteria is for the production of Yoghourt, and wherein said bacterium produces the contributive lactic acid of the peculiar taste of product.According to the production method of Yoghourt, the milk-acid bacteria living may reside in final sour-milk product.This type of existence of living bacterium is often considered to the feature of wanting in Yoghourt.For example, in Yoghourt, the existence of live lactobacillus is associated with some health and digestion benefit.Milk-acid bacteria, by the lactose fermentation in Ruzhong, makes the individuality with lactose intolerance be easier to digest Yoghourt.Human consumer is also associated other health advantages with existence that live and activated lactic acid bacteria culture in Yoghourt.Thereby think that it is that want and preferred by some human consumer institutes in Yoghourt, having the milk-acid bacteria living.
National Yogurt Association (N.Y.A.) requirement, Yoghourt has higher than the sum of bacterium in the activated lactic acid bacteria culture of a certain concentration and the active testing process of carrying out in the time that the shelf lives finishes during fabrication increases a logarithm, carrys out mark to contain strip of paper used for sealing alive and activated culture with explanation product.Food and Drug Association (F.D.A.) has proposed such regulation, its same bacterium that requires during fabrication and have a certain minimum in Yoghourt in the time that the shelf lives finishes, taking by product labelling as comprising alive and activated lactic acid bacteria culture.Thereby expect the milk-acid bacteria of the work existing in quantitative Yoghourt, to support this type of mark.
In other food, may not expect to exist high-caliber milk-acid bacteria.For example, in food, decline and can on food, produce adhesion coating as the hypertrophy of milk-acid bacteria in (deli style) sliced meat of Sa Ersa thick chilli sauce (salsa) product and prepared food type can cause tedious quality product.Although the hypertrophy of this bacterioid is harmless to human consumer, it does not cause appetite.Thereby, be desirably in by this type of product release to market before or the quantitative milk-acid bacteria of the further first being processed of this type of product composition.For example, if described product composition, as having unacceptable high-caliber bacterium for the preparation of the vegetables of Sa Ersa thick chilli sauce, can abandon these compositions so, thereby do not use these composition preparing products.Like this, in thering is the most at last the products production of unacceptable high-level bacterium and packing, can not consume extra investment.The meat product of prepared food type in manufacturing processed, be through sanitary measure and in the atmosphere of vacuum or improvement, pack; If this type of product stock in below 10 DEG C, expects that they can maintain high organoleptic's quality 2-4 week so.But, sometimes within the shelf lives, being occurred by microbial the becoming sour of lactic acid, this needs the producer to recall.Importantly can detect the milk-acid bacteria in prepared food meat product and reduce loss and product recall.
Traditionally, used selective medium as the milk-acid bacteria in the detection of the MRS of acidifying (de Man, Rogosa and Sharpe) agar quantitative food.But these class methods are consuming time, bacterial growth needs some days to reach the enough degree of bacterial colony visible on culture medium flat plate.This method is called dull and stereotyped cultivation, although useful, before acquisition result, needs a large amount of work and delay.During this period, food composition or product further process or render to market before possibly first keep.Thereby, expect to develop the fast method of the milk-acid bacteria in quantitative food, reduce the delay of processing and product release.
Method detects milk-acid bacteria with Bactometer faster.The food composition that Bactometer can produce as salad sauce with for Sa Ersa thick chilli sauce for detection of food is as the Bacterium lacticum (lactobacilli) of the vegetables of chopping.Method can comprise that product hatches 24 ± 1 hours containing in module hole Bacterium lacticum to substratum optionally at 30 ± 1 DEG C.Then described module is placed in to Bactometer system, and enumeration was associated with detection time.If any growth detected in module hole, material can be scoring on agar plate so, wherein on selective medium, after growth, can further identify described bacterium colony.But Bactometer carrys out quantitative bacterium by measuring the concrete physicochemical change being caused by bacterial growth.Because until growth occur just can bacterial detection, so the method is also consuming time.In addition, although some bacteriums are alive, cannot in substratum used, grow.These bacteriums are called alive but not can cultivate (VBNC) bacterium, and can not detect by Bactometer or cultural method.Thereby, expect the total milk-acid bacteria of exploitation in quantitative food fast and do not rely on the method for cultivation.
The method that is polymerase chain reaction for detection of a kind of technology that does not rely on cultivation of bacterial nucleic acid, is commonly referred to PCR, and it is generally considered to be for detection of the sensitiveest of the nucleic acid in given sample and method the most fast.In order to carry out PCR, need to be with a pair of oligonucleotide sequence of the nucleic acid complementation of bacterium as primer., a bacterial nucleic acid complementation for the 5' of primer and target bacteria nucleotide sequence end, and the bacterial nucleic acid complementation of the 3' of another primer and target nucleic acid sequence end.For the technology of PCR in real time (RT-PCR), use such probe, its target bacteria nucleic acid array complementation also and between two primers, allows to detect and quantitative objective material.
Although the technology of known PCR in real time can for detection of with quantitative objective nucleotide sequence, they depend on qualification unique sequences, to serve as primer and probe, and this qualification may be challenging.This type of sequence must be not only to target material uniqueness, but also can not be with himself in conjunction with forming primer dimer.Described probe also should meet design guidance policy, and the 5' end that is included in probe does not have guanine residue; There is suitable Tm value (melting temperature(Tm)); Short as much as possible, but at least there are 13 Nucleotide; Do not there is identical Nucleotide string (runs of identical nucleotides); And other features.Thereby the Article 1 of satisfied necessary these guilding principles of successful possibility for raising PCR in real time of qualification and Article 2 primer sequence and probe sequence may be difficult.
general introduction
The technology that embodiment of the present invention allow to use PCR in real time fast and specific detection and quantitative milk-acid bacteria.
Embodiment also comprises the test kit for detection of milk-acid bacteria, and it comprises Article 1 primer sequence, Article 2 primer sequence, and probe sequence, wherein the different zones complementation of the 16S rRNA gene of every sequence and lactic acid producing bacteria.For example, in some embodiments, the different zones complementation of each of Article 1 and Article 2 primer sequence and probe sequence and SEQ. ID. No. 4.
In some embodiments, comprise for the test kit of PCR detection milk-acid bacteria: the Article 1 primer that comprises SEQ. ID. No.1, the Article 2 primer that comprises SEQ. ID. No.2, and the probe that comprises SEQ. ID. No.3.
In some embodiments, probe comprises fluorophore, as Fluoresceincarboxylic acid.In some embodiments, test kit also comprises film opacity dyestuff.
In other embodiments, the present invention includes the method that detects milk-acid bacteria in sample, it comprises diluted sample, precipitate to produce centrifugal sample, use precipitation to carry out the extraction of DNA of bacteria, by extracted DNA of bacteria with comprise following PCR agent combination: the Article 1 primer that comprises SEQ. ID. No.1, the Article 2 primer that comprises SEQ. ID. No.2, the probe that comprises SEQ. ID. No.3, archaeal dna polymerase and deoxynucleotide triphosphoric acid, extraction DNA and the PCR reagent of combination are circulated by thermal cycler, and acquisition is corresponding to the data of the signal detecting by thermal cycler.Described method can also comprise the data corresponding to detected signal and typical curve are compared, with the amount of the lactic acid producing bacteria that exists in quantitative sample.In some embodiments, described sample is foodstuff samples, as Yoghourt.In some embodiments, probe comprises fluorophore, as Fluoresceincarboxylic acid.
accompanying drawing
Fig. 1 is the schema that detects the method for milk-acid bacteria according to embodiment of the present invention;
Fig. 2 is the PCR in real time result of sour-milk product;
Fig. 3 be in sour-milk product milk-acid bacteria with respect to the typical curve that uses the resultant Ct of PCR shown in Fig. 2;
Fig. 4 a is the PCR in real time result of Sa Ersa thick chilli sauce vegetables;
Fig. 4 b is the PCR in real time result of Sa Ersa thick chilli sauce vegetables;
Fig. 4 c is the PCR in real time result of Sa Ersa thick chilli sauce vegetables;
Fig. 5 a is the C of bacterial concentration with respect to the Sa Ersa thick chilli sauce vegetables shown in Fig. 4 a
ttypical curve;
Fig. 5 b is the C of bacterial concentration with respect to the Sa Ersa thick chilli sauce vegetables of the PCR result shown in use Fig. 4 b
ttypical curve; With
Fig. 5 c is the C of bacterial concentration with respect to the Sa Ersa thick chilli sauce vegetables of the PCR result shown in use Fig. 4 c
ttypical curve.
describe in detail
Have been found that now and can use PCR in real time qualification milk-acid bacteria.These milk-acid bacterias can be used and use round pcr optionally to increase to the special pair of primers sequence of these bacteriums, then can use also the special probe of these bacteriums is identified.Embodiment of the present invention can be used for the lactic acid producing bacteria in rapid detection and quantitative food.Exemplary food product for example comprises that sour-milk product, vegetables are if the meat product of the vegetables that produce for Sa Ersa thick chilli sauce and salting cucumber, prepared food type, salad sauce, tomato-sauce, milk-product are if cheese, fishery product are as catfish, wine, beer, lemonade, fruit juice and nectar (nectars).Embodiment of the present invention can also be used for environmental testing, as test foodstuff production equipment.
In Yoghourt, be found to be milk-acid bacteria alive and activated culture comprise thermophilus streptococcus (
streptococcus thermophilus), lactobacillus delbruockii subspecies bulgaricus (
lactobacillus delbrueckii subspecies bulgaricus) and Lactobacillus delbrueckii subsp. lactis (
lactobacillus delbruickii subspecies lactis).In the time that the technology of real-time quantitative PCR is applied to Yoghourt sample, can be fast and determine exactly the concentration (as the CFU of every gram of Yoghourt) of the colony-forming unit of these bacteriums.So, method of the present invention can be at specified time, as in the time that production completes (to support as described in mark that sour-milk product contains alive and activated culture) or in the time that the shelf lives finishes, and the quantity of the live lactobacillus quantitatively existing in sour-milk product.
Or embodiment of the present invention can be for quantitative vegetables, as the level of the lactic acid producing bacteria existing in the vegetables for the production of Sa Ersa thick chilli sauce or other food.For example, in Sa Ersa thick chilli sauce production process, first by vegetables chopping, then mix with sugar and salt and other compositions, be then placed in container as tank or vial, and in the end heat, kill potential pathogenic agent to carry out pasteurization.These vegetables can comprise tomato, onion, capsicum, corn, coriander etc.A kind of mode of monitoring Sa Ersa thick chilli sauce composition quality is the milk-acid bacteria existing in quantitative vegetables before pasteurization.As fruit vegetable has the milk-acid bacteria that is greater than threshold quantity, so may not can in the course of processing kill the milk-acid bacteria of q.s, thereby finished product may be destroyed by milk-acid bacteria, reduce shelf lives and quality product.These type of vegetables can abandon and cannot be used for Sa Ersa thick chilli sauce and produce.On the contrary, as fruit vegetable contains the milk-acid bacteria that is less than number of threshold values, can think that so further procedure of processing is enough to make final Sa Ersa thick-chilli sauce product not destroyed by milk-acid bacteria in normal shelf life.The threshold value of the vegetables of the chopping of producing for Sa Ersa thick chilli sauce in some embodiments, is 10
5cfu/ gram, and in other embodiments, threshold value is 10
6cfu/ gram.
In other embodiments, method and test kit for detection of with quantitative salad sauce in milk-acid bacteria.Basic detecting step is quantitatively similar to the detection of vegetables, comprises that sample preparation, DNA separate and RT-PCR reaction.
In other embodiments still, described method and test kit for detection of with the meat of quantitative prepared food type in milk-acid bacteria.Can comprise turkey, ham, roast beef, salami, chicken etc. for the example of the meat of the prepared food type of embodiment of the present invention.For fear of this situation, can test prepared food meat according to embodiment of the present invention, to detect with quantitative as the milk-acid bacteria in final prepared food meat product.
Embodiment of the present invention are used a pair of Oligonucleolide primers and probe, design its each with selectively targeted milk-acid bacteria.Primer pair comprise be positioned at the forward of sequence complementation of 5' end of target nucleic acid sequence or upstream primer and be positioned at target nucleic acid target sequence 3' end sequence complementation oppositely or downstream primer.Described target nucleic acid sequence is unique to milk-acid bacteria, and allows detected bacterium to be accredited as specifically milk-acid bacteria.In addition, primer pair and probe are special to target nucleic acid sequence, thereby they are not in conjunction with the nucleic acid of any other bacterium.
SEQ ID Nos. 1 and 2 shown in comprising below for the primer of embodiment of the present invention.The primer that is shown SEQ ID. No. 1 is above forward or upstream primer.The primer that is accredited as SEQ ID. No. 2 is reverse or downstream primer.Probe for embodiment of the present invention is below being shown SEQ ID. No. 3.In some embodiments, the probe that is accredited as SEQ ID. No. 3 is to have fluorophore and have the TaqMan probe of quencher at its 3' end at its 5' end.
PCR in real time probe can be used in embodiment of the present invention as TaqMan probe.The interior region complementation of the target oligonucleotide sequence between the target sequence region that can be combined for TaqMan probe and the primer of embodiment of the present invention.Before TaqMan probe and target sequence hybridization, the quencher of the fluorophore of probe one end and probe opposite end is geographically closely adjacent.In this state, quencher prevents that fluorophore from producing detectable signal.At each crossing phase of PCR circulation, probe starts to hybridize by the effect of archaeal dna polymerase with target sequence (or replication sequence of target sequence), in described process, probe and quencher are cut, thereby make no longer fluorophore described in quencher of their no longer closely adjacent and described quenchers.Therefore,, under hybridization state, fluorophore can detect.So, in each circulation, detected fluorescence level is directly related with the amount of the target nucleic acid sequence existing in sample.
Any real-time pcr fluorescence is rolled into a ball and all be can be used in embodiment of the present invention.For example, in some embodiments, described fluorophore is Fluoresceincarboxylic acid (FAM).Can use other fluorophores, as Tetrachlorofluorescein (TET).Can comprise tetramethyl-rhodamine (TAMRA) and dihydro cyclopyrrole diindyl (dihydrocyclopyrroloindole) tripeptides minor groove binding molecule for the example of the quencher of embodiment of the present invention.
The PCR in real time probe that can be used for the other types of embodiment of the present invention comprises equally SEQ. ID. No. 3 and can easily be identified by those skilled in the art.
The 16S rRNA gene complementation of each of serial ID Nos. 1,2 and 3 and milk-acid bacteria.These bacteriums comprise Lactobacillus delbrueckii subsp. lactis, lactobacillus delbruockii subspecies bulgaricus, thermophilus streptococcus, cunning leukonid (
leuconostoc fallax), benevolence sea leukonid (
leuconostoc inhae), hard leukonid (
leuconostoc gelidum), Argentinian leukonid (
leuconostoc argentium), leuconostoc mesenteroides sub species cremoris (
leuconostoc mesenteroides ssp cremoris), leuconostoc mesenteroides subsp mesenteroides (
leuconostoc mesentoides ssp mesenteroides),
leuconostoc gasicomitatum, pure lactobacillus (
leuconostoc kimchii), lemon leukonid (
leuconostoc cireum), pediococcus acidilactici (
pediococcus acidilactici) and Pediococcus pentosaceus (
pediococcus pentasaceus).In addition, because the 16S rRNA gene target of sequence is unique to milk-acid bacteria, each of primer and probe sequence will be only in conjunction with these bacteriums, make calling sequence have selectivity for qualification milk-acid bacteria.Embodiment of the present invention thereby comprise Article 1 and Article 2 primer and probe, its each all with 16S rRNA gene complementation.This allows fast and specific detection and quantitative milk-acid bacteria, and other bacteriums of error-detecting not.
Can provide the part of the 16S rRNA gene of the lactic acid of target being below shown as SEQ. ID. No. 4 for primer and probe sequence.
In some embodiments, Article 1 and Article 2 primer and probe separately with the different zones complementation of 16S rRNA gene, as the different zones of SEQ. ID. No. 4.For example, Article 1 primer can with first regional complementarity of SEQ. ID. No. 4, Article 2 primer can with second regional complementarity of SEQ. ID. No. 4, and probe can with the 3rd of SEQ. ID. No. 4 regional complementarity, wherein first, second or the 3rd region do not have overlapping.
Detect with the method for quantitative lactic acid bacterium number and be shown in the schema that is designated Fig. 1 according to embodiment of the present invention.
First step is the preparation 10 of test sample.Described test sample is such material, as has the food of unknown quantity bacterium.For test sample, as Yoghourt, preparation can be included in solution as dilute sample in damping fluid (as buffered peptone water).Or, sample can be diluted in water or other suitable liquid.For test sample, as Sa Ersa thick chilli sauce vegetables and prepared food meat, preparation can comprise sample and liquid are mixed as damping fluid or water, and then sucking liquid, uses the sample of described liquid as further test subsequently.
Next step is centrifugal sample 20.Then by centrifugal the liquid of diluted Yoghourt or sucking-off (for example, for Sa Ersa thick chilli sauce vegetables and prepared food meat), to isolate bacterium from liquid.Then the precipitation by centrifugal acquisition is tested for further.
Next step is that DNA separates 30(also referred to as DNA extraction) so that described DNA is easy to use PCR to copy and detect.DNA separates 20 and comprises broken bacteria cell wall, to discharge genomic dna.For example, this can pass through physics fracture cell walls, as by mixing with granulated glass sphere, or has broken by chemistry or enzymatic.Then, remove protein and fatty soluble compound, for example, as passed through to use suitable chemical, as phenol or ethanol.Finally, for example, as passed through filtration and the wash-out of DNA, or other technologies well known by persons skilled in the art are come combination, extraction and concentration DNA.Existence can be used for several commercial kits that DNA of bacteria separates.For example, DNA test kit can be bought from Norgen Biotek Corp., Zygen, Promega, Qiagen, Mo Bio Laboratories and other.A kind of this type of test kit is can be from Mo Bio Laboratories, the PowerFood Microbial DNA Isolation Kit that Inc obtains.
Next step is preparation feedback mixture 40.Reaction mixture can comprise the DNA of primer, probe and extraction, and PCR reagent.Described PCR reagent can comprise archaeal dna polymerase, as Taq polysaccharase, MgCl
2, dNTPs(VITAMIN B4, thymus pyrimidine (Thiamine), cytosine(Cyt) (Cytosing) and guanine) and water.Can buy and obtain several commercial PCR in real time reagent, it comprises these components, as the test kit of Bio-Rad, Quanta Bioscience, Qiagen, Invitrogen and other productions.Reaction mixture can be prepared by the reacting hole composite reaction thing of the RT-PCR Sptting plate for by the processing of PCR thermal cycler.
Then,, as optional step, can seal the hole of containing prepared reaction mixture centrifugal under the condition to remove any bubble from hole.Described optional step can be preferably individual by some, but optional.
Once reaction mixture is ready, Sptting plate is inserted into and in RT-PCR system, carries out thermal cycling 40.In PCR thermal cycling step, target genetic stocks the gene copy number increase is to the level that can detect and can be quantitative.This step can be used any RT-PCR thermal cycler to carry out in any RT-PCR system.Described RT-PCR system can comprise thermal cycler, for fluorescence excitation and transmitting collect optical element and there is data acquisition and the computer of analysis software.For example, real-time PCR system can obtain from Applied Biosystems and Bio-Rad by commercial sources.
Next step is to detect the probe 50 that can exist after each circulation of thermal cycler.In PCR process, any fluorescence being produced by fluorescence dye is detected by RT-PCR system.Along with the gene copy number increase of target sequence, the amount of fluorescence increases with each circulation.It is cycle threshold or Ct that fluorescence reaches the above needed cycle number of fluorescence threshold.Fluorescence threshold is that it can be adjusted for each experiment, thereby makes in the region of its exponential amplification on all curves according to any level of the fluorescence of baseline value selection.The sample with larger quantity bacterium will reach fluorescence threshold quickly, and thereby there is lower Ct value, and those samples with bacterium still less by more needs circulations to reach fluorescence threshold, and thereby there is higher Ct value.Thereby Ct value can be to determine the useful basis of the bacterial concentration of test sample.But the data that obtain from RT-PCR system, if fluorescence data is as Ct value, must compare for known standard, so that PCR data are associated with corresponding bacterial concentration.
Thereby next step is the conversion of the respective value of RT-PCR data and bacterial concentration.In some embodiments, this comprises that comparison RT-PCR data and known standard are as typical curve.In some embodiments, the RT-PCR data that use are Ct values, and typical curve is the curve of bacterial concentration than Ct.This type of known standard can with the same terms of the test sample of for example below describing in embodiment 1 and 2 under on the sample of known bacterial concentration, carry out RT-PCR and prepare by having.In other embodiments, known standard of comparison can be provided as mathematical formula (as the formula of the line for typical curve), and the respective value that RT-PCR data are converted into bacterial concentration can be comprised the RT-PCR data of unknown sample (as Ct value) are put into mathematical formula, to obtain the value of bacterial concentration.In other embodiments still, known standard of comparison can be question blank, and can be by RT-PCR data and described table comparison, to determine the respective value of bacterial concentration.
RT-PCR technology have detect live with dead biology in the ability of the DNA that exists.But, for many objects, only interested in the milk-acid bacteria living.For example, in the time of the quantity of the milk-acid bacteria that embodiment of the present invention are existed for quantitative Yoghourt, expect only to detect live and activated bacterium for Yoghourt is labeled as and contains alive and activated culture.Equally, food as prepared food meat and Sa Ersa thick chilli sauce (wherein not wishing the existence of lactic acid producing bacteria) in, only close the bacterium of being indecisive and changeable, this is because the hypertrophy of described bacterium causes the reduction of food quality, and this hypertrophy only just occurs when bacterium is to live.
In optional embodiment, method and system can be for environmental testing and monitoring.For example can from environment, obtain sample by carrying out wiping machine surface with the wiper of sponge, swab or other types.In some embodiments, environment sponge sampling test kit can be for sampling from environment.Then the substratum of enrichment can be joined in sponge test kit for pre-concentration and the felicity condition of growth as 36 DEG C at overnight incubation.Then by centrifugal come concentrating cells, be subsequently as described herein DNA separate and RT-PCR react.
In some embodiments, can test specific acid dairy products twice, grow with alleged occurrence.Because only bacterium alive just can grow, therefore detected bacterium of accelerating must be alive.For example, can carry out the PCR detection method of milk-acid bacteria in sour-milk product, then can after like products is hatched 4 hours at 43 DEG C, carry out again repetition.Can think hatch rear milk-acid bacteria increase (as log increase) representative live bacterium or due to live bacterium, this be because only live bacterium just can grow.
Or additive method can be for ensureing that all milk-acid bacterias that use embodiment as herein described to detect are alive.In some embodiments, film opacity dyestuff can be optionally for before extracting DNA.This type of dyestuff only reacts in conjunction with the DNA of dead cell and the RT-PCR that this type of DNA can not be used for subsequently.The example of this type of film opacity dyestuff comprises single nitrine second ingot (EMA) and single folded aziridine (PMA).In other embodiments, first isolation of RNA from test sample of described method, instead of DNA.Different from DNA, once bacterium death, RNA degraded is very fast, and the amount of the RNA existing in sample is directly related with the bacterial count of living.Then can be by RNA and reversed transcriptive enzyme combination, so that rna transcription is become to DNA.Therefore the DNA producing then can be for using primer as herein described and probe to carry out RT-PCR.
Embodiment of the present invention comprise the method for detection and quantitative milk-acid bacteria, and also comprise the test kit for using RT-PCR detection and quantitative milk-acid bacteria.In some embodiments, described test kit comprises Article 1 primer and Article 2 primer and probe, and it is all complementary and special to it with milk-acid bacteria.In some embodiments, the target sequence of primer and probe is in the 16S of milk-acid bacteria rna gene.In some embodiments, primer comprises SEQ ID Nos. 1 and 2, and probe comprises SEQ ID No. 3.Described probe can comprise fluorescent mark and can be TaqMan probe.In some embodiments, described test kit can also comprise one or more archaeal dna polymerases, dNTP's, water and/or magnesium chloride.In some embodiments, described test kit further comprises film opacity dyestuff.
experiment
In following examples, the PerfecCTA qPCR FastMix buying from Quanta Bioscience is used as to PCR reagent.The MiniOpticon buying from Bio Rad is used as to RT-PCR thermal cycler.Manufacture SEQ ID Nos. 1 and 2 as RT-PCR primer by Applied Biosystems, and manufacture serial ID No. 3 for thering is the probe of 6-Fluoresceincarboxylic acid mark, and these primers and probe are for each embodiment.
Embodiment 1
In this embodiment, the relative concentration that produces the milk-acid bacteria existing in sour-milk product is in the typical curve of Ct.By 12 kinds of sour-milk product for generation of typical curve.Be 10 by concentration
5the milk-acid bacteria of cfu/ gram is for the preparation of Yoghourt.In the fermenting process of 4 hours, per hourly from fermentor tank, sample Yoghourt once.Independently to three fermentor tank samplings.As described below by cultivating sour-milk product and count bacterial cultures and determine bacterial concentration at M17 and the dull and stereotyped upper flat plate of MRS.In addition, use RT-PCR to test identical sour-milk product, also as further discussed below.Then by cultivating definite bacterial concentration by flat board to the mapping of RT-PCR result, to produce typical curve, it can determine the bacterial concentration in this sample for the RT-PCR result based on the unknown test Yoghourt sample.
The dull and stereotyped Yoghourt of cultivating carries out as follows to determine bacterial concentration.Bacteria levels based on prediction is by difference amount dilution Yoghourt, to obtain the necessary enumeration between 25-255 bacterium colony on flat board.Find 12 kinds of sour-milk product have fall into 4 roughly grouping bacteria levels: 10
5, 10
7, 10
8with 10
9cFU/g.
In order to prepare sour-milk product for using the test of RT-PCR, 11 grams of Yoghourt samples are mixed with 99 ml buffered peptone waters, to form the dilution of 1:10.The Yoghourt sample that 1ml is diluted joins in 2ml pipe and at 13000 g, within RT centrifugal 5 minutes, precipitates to form.Once will precipitate for further test, pour out supernatant liquor.Then, use from MO BIO Laboratories, the PowerFood Microbial DNA Isolation Kit that Inc. buys extracts DNA of bacteria according to the following description of product from each precipitated product.Described cell precipitation is resuspended in the solution PF1 of 450ul.The cell of resuspension is transferred in Microbead pipe and vortex 10 minutes, then in Microbead pipe at 13000 g, under RT centrifugal 1 minute.Supernatant liquor is transferred in the pipe with 100ul solution PF2, and vortex is to mix, and then at 4 DEG C, hatches 5 minutes.Then by it in PF2 pipe at 13000 g, under RT centrifugal 1 minute, then the solution PF3 of 900ul is mixed in clean pipe with supernatant liquor.Then the PF3 of 650ul and sample mix solution are loaded in vacuum rotating filter pipe, and repeat this process until all supernatant liquors are all loaded in rotary filter.Then add the solution PF4 of 650ul, add subsequently the solution PF5 of 650ul.Pipe, at 13000 g, under RT centrifugal 2 minutes, is then placed in rotary filter basket to clean 2ml pipe, and adds the solution PF6 of 100ul.By pipe at 13000 g, under RT centrifugal 1 minute, and abandon rotary filter basket.Then DNA can be for RT-PCR or freezing or refrigerate and store.
Carrying out in the set-up procedure of RT-PCR, by PCR reagent, primer, probe and the water combination of the amount to show in table 1 are below carried out to the pre-composition (Master Mix) for the preparation of each sample.To each plan totally three repetition of 12 kinds of Yoghourt samples, amount to 36 reactions, thereby prepared the pre-composition (to ensure that to 36 reactions of plan be enough) of the q.s for amounting to 38 reactions.
Table 1:
| Reagent | The volume (μ L) of each reaction needed | The cumulative volume (μ L) that total overall reaction needs |
| PERFECTA QPCR FASTMIX | 10 | 380 |
| Primer and probe mixture | 1 | 38 |
| dH2O | 7 | 266 |
| The DNA extracting | 2 | 76 |
| Amount to | 20 | 760 |
Then,, for each reaction, the DNA of 18 μ l pre-compositions of as above preparation and 2 μ l extraction is combined in the hole of RT-PCR Sptting plate.Each the every a DNA of extraction preparation of samples from 12 kinds of sour-milk product is repeated for 3 times totally, amount to 36 reactions.Sealed reaction plate under 25000 rpm centrifugal 2 minutes.
Sptting plate is inserted in thermal cycler and is processed as shown in below table 2.
Table 2
| Temperature | Time length | Quantity |
| 95℃ | 10 minutes | Once |
| 95℃ | 15 seconds | 40 circulations |
| 60℃ | 1 minute | 40 circulations |
| Signal-obtaining (signal readying) | (after each circulation) | 40 circulations |
In PCR process, obtain fluorescence reading from each reaction.Fluorescence (RFu) is shown in Fig. 2 with respect to the curve of circulation.PCR cycle number when Ct is defined as to reporter molecule fluorescence and is greater than threshold value, described threshold value is any level of the fluorescence selected according to baseline variability.
The Ct value that three of each sour-milk product are repeated to obtain is averaged, and these average results with cultivate definite corresponding bacterial count by flat board and be shown in table 3 below.Finally, for the curve of Ct with respect to bacterial concentration (log CFU/ gram) drawn in each reaction shown in Fig. 3, to produce typical curve.
Table 3
。
Embodiment 2
In this embodiment, Sa Ersa thick chilli sauce vegetables are drawn the typical curve of milk-acid bacteria concentration.By three groups of Sa Ersa thick chilli sauce vegetables (as for Sa Ersa thick chilli sauce produce) for the preparation of typical curve.With the milk-acid bacteria of three kinds of different levelss (be probably 10
8cfu/ gram, 10
6cfu/ gram and 10
4cfu/ gram) to the puncture of Sa Ersa thick chilli sauce vegetables group.Test each time in triplicate, to adapt to biological variability, and then repeat described test.Identical with the Yoghourt typical curve in embodiment 1, by cultivating at M17 and MRS upper flat plate the bacterial concentration of determining in Sa Ersa thick chilli sauce vegetables.Bacteria levels based on prediction is diluted described Sa Ersa thick chilli sauce vegetables by difference amount, to obtain the necessary enumeration between 25-255 bacterium colony on flat board.Obtain corresponding RT-PCR data, with to Sa Ersa thick chilli sauce vegetables drawing standard curve.
In order to prepare Sa Ersa thick chilli sauce vegetables for RT-PCR, the Sa Ersa thick chilli sauce vegetable sample of 11 grams of bacteriums puncture is mixed with 99ml buffered peptone water, to form 1:10(a on to ten) dilution.1ml dilute sample is joined in 2ml pipe, and at 13000 g under RT centrifugal 5 minutes.Pour out supernatant liquor and remaining precipitation is separated for DNA.
Then use the Laboratories from MO BIO, the PowerFood Microbial DNA Isolation Kit that Inc. buys extracts DNA of bacteria according to the description of product of describing in embodiment 1 from Sa Ersa thick chilli sauce vegetables precipitation.
By PCR reagent, primer, probe and cDNA as shown in below table 4 are combined to prepare pre-composition, react for RT-PCR.Every kind of Sa Ersa thick-chilli sauce product of the triplicate test of plan, thereby prepared the enough pre-compositions for 10 reactions, thus at least 9 reactions each time of twice test of operation.
Table 4
| Reagent | (μ l) for the volume of each reaction needed | (μ l) for the cumulative volume that total overall reaction needs |
| PERFECTA QPCR FASTMIX | 10 | 100 |
| Primer and probe mixture | 1 | 10 |
| The DNA extracting | 9 | 90 |
| Amount to | 20 | 200 |
For each reaction, the DNA of 11 μ l pre-compositions and 9 μ l extraction is combined in the reacting hole of RT-PCR Sptting plate.For each Sa Ersa thick chilli sauce sample, react in triplicate.Sealed reaction hole under 25000 rpm centrifugal 2 minutes subsequently.After centrifugal, reacting hole is inserted in thermal cycler, and carry out RT-PCR according to parameter as shown in table 5.
Table 5
| Temperature | Time length | Quantity |
| 95C | 10 minutes | Once |
| 95C | 15 seconds | 40 circulations |
| 60C | 1 minute | 40 circulations |
| Signal-obtaining (signal readying) | (after each circulation) | 40 circulations |
In RT-PCR process, obtain fluorescence reading.The RT-PCR data of all samples in Fig. 4 a, 4b and 4c, are shown.Determine the Ct value of each sample based on threshold fluorescence value.The Ct value of each group of three duplicate samples of each Sa Ersa thick chilli sauce vegetables group is averaged, and the respective value of averaging of income Ct and bacterial concentration (Log CFU/g Sa Ersa thick chilli sauce) is shown in below in table 6.Average Ct value is mapped to bacterial concentration (Log CFU/g Sa Ersa thick chilli sauce), to produce the typical curve of the Sa Ersa thick chilli sauce vegetables shown in Fig. 5 a, 5b and 5c.Can find out, the result of first group and second group test is practically identical, has confirmed the reliability of testing method.
Table 6:
。
Claims (21)
1. the test kit that detects milk-acid bacteria for PCR, it comprises:
The Article 1 primer that comprises SEQ. ID. No. 1;
The Article 2 primer that comprises SEQ. ID. No. 2; With
The probe that comprises SEQ. ID. No. 3.
2. the test kit of claim 1, wherein said probe further comprises fluorophore.
3. the test kit of claim 2, wherein said fluorophore comprises Fluoresceincarboxylic acid.
4. the test kit of claim 2, it further comprises film opacity dyestuff.
5. for detection of the test kit of lactic acid producing bacteria, it comprises:
Article 1 primer with first regional complementarity of SEQ. ID. No. 4;
Article 2 primer with second regional complementarity of SEQ. ID. No. 4;
Probe with the 3rd regional complementarity of SEQ. ID. No. 4.
6. the test kit of claim 5, wherein said Article 1 primer comprises SEQ. ID. No. 1.
7. the test kit of claim 5, wherein said Article 2 primer comprises SEQ. ID. No. 2.
8. the test kit of claim 5, wherein said probe comprises SEQ. ID. No. 3.
9. the test kit of claim 8, wherein said probe further comprises fluorophore.
10. the test kit of claim 9, wherein said fluorophore comprises Fluoresceincarboxylic acid.
11. test kits for PCR detection lactic acid producing bacteria, it comprises:
Article 1 primer with first regional complementarity of the 16S rRNA gene of milk-acid bacteria;
Article 2 primer with second regional complementarity of the 16S rRNA gene of milk-acid bacteria;
With the 3rd regional complementarity of the 16S rRNA gene of milk-acid bacteria and be marked with the probe of fluorophore.
The test kit of 12. claims 11, wherein said Article 1 primer comprises SEQ. ID. No. 1.
The test kit of 13. claims 11, wherein said Article 2 primer comprises SEQ. ID. No. 2.
The test kit of 14. claims 11, wherein said probe comprises SEQ. ID. No. 3.
The test kit of 15. claims 14, wherein said probe further comprises fluorophore.
The test kit of 16. claims 15, wherein said fluorophore comprises Fluoresceincarboxylic acid.
17. detect the method for milk-acid bacteria in sample, and it comprises:
Prepare described sample;
Use the sample of preparation to carry out DNA of bacteria extraction, to obtain the DNA of bacteria of extraction;
By the DNA of bacteria of described extraction and PCR agent combination, the Article 1 primer that described PCR pack contains SEQ. ID. No. 1, the Article 2 primer that contains SEQ. ID. No. 2, the probe that contains SEQ. ID. No. 3, archaeal dna polymerase and deoxynucleotide triphosphoric acid;
DNA and the PCR reagent of the extraction after combination are circulated by thermal cycler; With
Obtain the data corresponding to the signal detecting by described thermal cycler.
The method of 18. claims 17, it further comprises the data of the described signal corresponding to detecting and typical curve is compared, with the amount of the lactic acid producing bacteria that exists in quantitative described sample.
The method of 19. claims 18, wherein foodstuff samples comprises Yoghourt.
The method of 20. claims 17, wherein said probe further comprises fluorophore.
The method of 21. claims 20, wherein said fluorophore comprises Fluoresceincarboxylic acid.
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| US201161501470P | 2011-06-27 | 2011-06-27 | |
| US61/501470 | 2011-06-27 | ||
| PCT/US2012/043958 WO2013003262A1 (en) | 2011-06-27 | 2012-06-25 | Detection and quantification of lactic acid producing bacteria in food products |
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| KR (1) | KR20140057246A (en) |
| CN (1) | CN103930564A (en) |
| AU (1) | AU2012275650A1 (en) |
| CA (1) | CA2840364A1 (en) |
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| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
| CN113817810A (en) * | 2021-08-09 | 2021-12-21 | 宁波大学 | Novel qPCR (quantitative polymerase chain reaction) method for detecting lactic acid bacteria in fermented milk |
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| IT201600097963A1 (en) * | 2016-09-29 | 2018-03-29 | Univ Degli Studi Di Bari Aldo Moro | Molecular method aimed at authenticating traditional / typical bread obtained with natural yeast, primers used in the said method and kit containing said primers. |
| EP3774835A4 (en) * | 2018-04-13 | 2022-01-19 | National Agricultural Genotyping Center | Universal lactic acid bacteria quantification kits, methods, compositions and apparatuses therefor |
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| US6403468B1 (en) * | 1998-08-20 | 2002-06-11 | Nec Corporation | Method for forming embedded metal wiring |
| US7309589B2 (en) * | 2004-08-20 | 2007-12-18 | Vironix Llc | Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing |
| US20070292852A1 (en) * | 2004-02-17 | 2007-12-20 | Sapporo Breweries Limited | Method of Detecting and Identifying Lactic Acid Bacterium |
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| US6103468A (en) * | 1997-10-07 | 2000-08-15 | Labatt Brewing Company Limited | Rapid two-stage polymerase chain reaction method for detection of lactic acid bacteria in beer |
| JP4813215B2 (en) * | 2005-03-08 | 2011-11-09 | 三星電子株式会社 | Probe set design method, probe set designed thereby, microarray including the probe set, computer-readable recording medium recording a program enabling a computer to execute the method, and target sequence using the probe set Identification method |
| JP2006333801A (en) * | 2005-06-03 | 2006-12-14 | Obihiro Univ Of Agriculture & Veterinary Medicine | Method for detecting brucella canis and leptospira and primer set used therefor |
| WO2007106407A2 (en) * | 2006-03-10 | 2007-09-20 | Wyeth | Microarray for monitoring gene expression in multiple strains of streptococcus pneumoniae |
| FR2908785B1 (en) * | 2006-11-17 | 2012-09-28 | Air Liquide | MICROORGANISM DETECTION METHODS BY PCR |
-
2012
- 2012-06-25 AU AU2012275650A patent/AU2012275650A1/en not_active Abandoned
- 2012-06-25 MX MX2014000078A patent/MX2014000078A/en unknown
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- 2012-06-25 WO PCT/US2012/043958 patent/WO2013003262A1/en active Application Filing
- 2012-06-25 EP EP12803677.9A patent/EP2723903A4/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403468B1 (en) * | 1998-08-20 | 2002-06-11 | Nec Corporation | Method for forming embedded metal wiring |
| US20070292852A1 (en) * | 2004-02-17 | 2007-12-20 | Sapporo Breweries Limited | Method of Detecting and Identifying Lactic Acid Bacterium |
| US7309589B2 (en) * | 2004-08-20 | 2007-12-18 | Vironix Llc | Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
| CN107937581B (en) * | 2017-12-28 | 2021-09-17 | 内蒙古农业大学 | Amplification primer pair for lactobacillus sequencing, lactobacillus species identification method and application |
| CN113817810A (en) * | 2021-08-09 | 2021-12-21 | 宁波大学 | Novel qPCR (quantitative polymerase chain reaction) method for detecting lactic acid bacteria in fermented milk |
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| WO2013003262A1 (en) | 2013-01-03 |
| MX2014000078A (en) | 2014-05-01 |
| EP2723903A4 (en) | 2015-02-18 |
| KR20140057246A (en) | 2014-05-12 |
| CA2840364A1 (en) | 2013-01-03 |
| WO2013003262A9 (en) | 2014-02-13 |
| EP2723903A1 (en) | 2014-04-30 |
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