WO1996024685A1 - Process for obtaining specific probes of strain or specie - Google Patents

Abstract

The invention provides for the rapid and simple selection of fragments of DNA amplified by a non specific PCR reaction, fragments which are then used as specific probes of strain or specie, as desired. The fragments extracted from an agarose gel will be used as a probe in a hybridation on a solid medium whereon DNA samples have been immobilized, whether amplified or not, of bacterial strains to be identified. With the disclosed process it is possible to obtain specific probes applicable to the rapid identification of species in various fields.

Description

PROCEDURE TO OBTAIN SPECIFIC STRAP OR SPECIES PROBES

BACKGROUND Here we describe a quick and simple method for the identification of strains and bacterial species or other organisms by one or two successive steps, one of synthesis (random amplification) of DNA and another of membrane hybridization. The identification of bacterial species of industrial and clinical interest requires, in many cases, several days from when the colonies are visualized on selective medium until they know the fermentation pattern of sugars and other physiological characteristics of the species. Frequently its antigenic pattern or phage sensitivity (typing) should be determined, as occurs in Escherichia coli, Salmonella spp. and large number of pathogenic species. In the case of lactic bacteria, dichotomous keys offer the best alternative to quickly and efficiently identify new isolates (Montel,. -C., Heel, R. Fournaud, J. and Champomier, -C., 1991, J Appl. Bacteriol. 79: .469-472; Shillinger, V. And L. cke, F. -K., 1987, Food Microbiol. 4: 199-208). However, a series of costly determinations in time and means are still needed.

Molecular techniques have recently been developed to make the bacterial identification process faster and more reliable. These techniques are: (i) solid support hybridization of species-specific probes (cloned genes or synthetic oligonucleotides); (ii) restriction pattern polymorphism in agarose gels, generally followed by hybridization with probes of the rRNA genes (ribotyping); (iii) an important variant of this is the determination of the restriction pattern by electrophoresis in pulsed field gels; (iv) and the determination of the protein electrophoresis pattern in acrylamide gels. Of all the mentioned techniques, the hybridization with own probes of species of a higher level of "specificity" and "sensitivity" (understood as specific a low number of false positives, and by sensitivity, little false negatives). However, it requires a long previ work for cloning, selection of species-specific fragments and even sequencing.

The chain polymerization reaction (PCR) is, in quantitative terms, the technique that has generated the greatest number of research work in recent years for the identification of both clinical isolates and species of interest in food technology. There are two variants of the technique, namely: (i) the amplification of fragments of A corresponding to known (specific) genes, where oligonucleotides used as primers for the reaction synthesis in PCR only recognize a specific region of the bacterial gene (BUBERT , A., KOHLER, S., and GOEBEL, W., 1992, App Environ. Microbiol. 58: 2625-2632; HERMÁN, L., and DE RIDDER, H. 1992, Appl. Environ. Microbiol. 58: 2099 -2101; JENSEN, MA WEBSTER, JA, and STRAUS, N., 1993, Appl. Environ. Microbiol. 5 945-952; KLIJN, N., WEERKAMP, AH, and DE VOS, WM, 199 Appl. Environ. Microbiol 57: 3390-3393; TJHIE, HT. R00SENDAAL, R., WALBOOMERS, JMM, THEUNISSEN, JJH, TJON L SANG, RRM, MEIJER, CJLM, MacLAREN, DM and van d BRULE, AJC, 1993, J. Microbiol. Methods 18: 137-15 VILARO, M., JAULAC, B., RIFAI, S., NICOLINI, P., PIEMONT, Y. M0NTEIL, H., 1993, J. Microbiol. Methods 18: 83-90; WERNARS, K. HE W ELMAN, K., NOTERMANS, S., DOMANN, E., LEIMEISTER-WACHTE M., and CHAKRABORTY, T., 1992, Appl. Environ. Mi crobiol 5 765-768); and (ii) non-specific amplification with a universal primer, which initiates DNA synthesis at different locations of the genome, and which gives rise to what is called DNA polymorphism by random amplification (RAPD = rand amplification polymorphism of DNA), using for the strain of strains (CANCILLA, MR, POWELL, IB, HILLIER, AJ, DAVIDSON, BE, 1992 Appl. Environ. Microbiol. 58: 1772-177 CZAJCA, J., BSAT, N., PIANI, M., RUSS, W., SULATANA, K., WIEDMANN, M., WHITAKER, R., and BATT, CA, 1993, Appl. Environ.Microbiol. 58: 2948-2953; HAMELIN, RC, OUELLETTE, GB, and BERNIER, L., 1993, Appl. Environ. Microbiol 59: 1752-1755; MAZURIER, S.-I., ANDURIER, A., MARQUET-VAN DER MEER, N., NORTHERMANS, S., and WERNARS, K., 1992, Res. Microbiol. 143: 507-512; RALPH, D., MCCLELLAND, M., WELSCH, J., BARANTON, and G., PEROLOT, P., 1993 J. Bacteriol. 175: 973-981; ROSS, BC, and DWYER, B., 1993, J. Clinical Microbiol. 31: 329-334). Both techniques have advantages and disadvantages. Amplification with known gene fragments has great "sensitivity" and good "specificity", but requires a deep genetic knowledge of the species. On the other hand, random fragment amplification (RAPD) is economical in material media and usually gives good results to discriminate different strains and species with the same or the same oligonucleotides; However, this technique is not applicable for a direct identification of new species, since the pattern of bands observed is sometimes variable from one experiment to another. But even in these cases, there is always one or more bands of DNA that are conserved within the same species. The technique described is based precisely on the isolation of these bands from the agarose gel and its use as a probe to recognize the strains that belong to the same species. On the contrary, unique bands from one line may also be used to recognize it among others of the same species.

Using the technique proposed here, DNA fragments amplified by a nonspecific PCR reaction are quickly and easily selected, which are then used as specific probes of lineage or species, as chosen. The fragments extracted from an agarose gel will be used as a probe in a hybridization on solid support in which samples of DNA, amplified or not, of bacterial strains to be identified have been immobilized. The method is still more effective if there is a rapid bacterial cell lysis technique, as described in the example.

In this way, the great limitation of the methods based on nucleic acid hybridization is overcome, although they are more sensitive and specific, they need to obtain a sufficiently specific DNA probe, which entails a long period of work. .

DESCRIPTION OF THE TECHNIQUE

The method consists of four phases:

1) Random amplification of DNA fragments with the chain polymerization reaction (PCR) in different strains of the same species. It is convenient to analyze as many strains as possible, for which it is very useful to have a quick lysis method and compatible with the application of the PCR. Once the DNA of the different samples is amplified, an electrophoresis is performed to separate the amplified fragments by sizes.

2) Selection and isolation of DNA fragments of one or several sizes, and that are present in all samples of the same species, if it is desired to have a specific probe d species. You can also select, from one line, missing fragments in the other samples of lineages of a given species, which will give you a specific lineage probe.

3) The strips removed are marked by preferably non-radioactive AD marking methods, so that the selected fragments (one or more per species) can be used as a probe in the next step of the technique. 4) Detection of those samples that have DNA homologous to the probe selected from a set of problem samples. To do this, nucleic acids from used cell phones, amplified by PCR or not, will be fixed to nitrocellulose nylon membranes, or to any solid support. Then, it will undergo washing and the relevant treatments to hybridize with the previously prepared probes. The hybridized probe will be detected by one of several methods available, which are a function of the method of tide used and the solid support to which the samples are fixed, such as exposure to a photographic film, colorimetric, fluorimetric or aggregation methods. antibodies The positives will be resolved as belonging to the species or line to which the probe belongs.

This procedure allows, first to select species-specific probes in one or two weeks, with the hybridization tests necessary to verify the specificity of the selected fragments. Secondly, once a probe is obtained, the identification of species, from colonies, can be done in an average time of 48 hours.

If there is also an effective method of rapid lysis from bacterial cells from agar colonies, it is possible to shorten the time needed to obtain probes by about 5 days, and by half the time required for identification tests (24h ).

An example is described below that only illustrates its possible applications.

EXAMPLE 1.- Among the molecular methods currently used for the identification and typing of bacterial species, is the so-called RAPD, abbreviation of the Saxon terms of "polymorphism of DNA fragments by random amplification. "In this technique, one uses several oligonucleotide primers that fix the onset of DNA synthesis by a nonspecific thermophilic polymerase. The number of denaturation, ringing and synthesis cycles may vary, but they are generally used in around 30. Several factors are important when it comes to getting a reproducible pattern, for example, the slope of thermal recovery between the different cycles, the temperature and time of banding, concentration of Mg + 2 ions, etc. The method here It has been developed with purified AD from type strains of Lactobacillu species usually found in meat products.In addition, a rapid cell lysis technique was developed from d colonies on MRS agar plates that significantly abbreviated e process and makes it applicable to a large number of species of lactic bacteria. For rapid lysis, a colony of lactobacilli is touched with a stick or, by depositing the adhered cells in an eppendorf tube containing 50 ml of lysis buffer (1 mM TRIS.C1H, pH 9 * 0; O'L% TritonxlOO) with 10 mg / ml lysozyme and 80 U / ml mutanolisin. The eppendorf tubes are then incubated 30 minutes at 372 C and the cells are used to bring the tubes to a boil for 5 minutes. With 1 ml of each tube and sufficient for amplification reactions. Then, the chain polymerization reaction (PCR) is carried out, adding for each reaction: 1 ml of bacterial DNA (O'l if it has been previously purified), 50 pmol of the oligonucleotide designed for this purpose (e.g., 5'-GGATCCAAGACAACGTTTCAAA -3 '), 5 pmol dNTP, 1.5 mM MgC12, Taq buffer and 0.5 U Taq polymers (Boehringer Mannheim). The banding temperature in the first two cycles is 45QC and in the next 30 cycles of 52 ° A electrophoresis of 1% agar gel is then carried out and the bands are visualized in EtBr.

With this procedure amplification patterns have been obtained in a number of species of lactic bacteria (Figure 1) from its DNA extracted and purified by conventional methods and by the rapid method described (Figure 2). It can thus be observed that the amplification pattern is different and proper for each of the species analyzed. In these electrophoresis and in those of Figure 3, it is also observed that within the same species (see the case of Lactobacillus plantarum and Lactobacillus casei subsp.casei) a number of bands are conserved, while others of each of each of the lineages of the species.

These fragments of the same size that are conserved within the members of a species, are extracted from the agarose with Sephaglass (Pharmacia Biotechnology, Uppsala, Sweden), or another known procedure, and proceed to its marking with digoxigenin (eg. with the nick translation method of Boehringer Mannheim, Mannheim, Germany). These fragments will serve as a probe in subsequent hybridizations. On the other hand, the DNA extracted by the rapid method of different lactobacilli and amplified by PCR is absorbed into a nylon membrane, to be hybridized to the prepared probes. Subsequently, an anti-digoxigenin antibody, a phosphatase conjugate and the chemiluminescence of the AMPPD is used for development. It can be seen (Figure 4), that one of the probes selected from Lb. curvatus hybrid only with the amplified DNA of this species, not hybridizing with the DNA of Lb. casei, Lb. sake or Lb. plantarum (not seeing dark spot after exposure and development of the photographic film). The same goes for the specific probes of Lb. sake or Lb. plantarum The amount of purified non-amplified DNA fixed to the membrane (about 20 ng) seems to be at the resolution limit of the method.

The results show that after brief hybridization tests, highly species-specific probes can be selected in a simple way. DESCRIPTION OF THE FIGURES

FIGURE 1A.- Bands amplified by the DNA polymerization reaction carried out on DNA (purified by the method of Marraur, 1961. J. Mol. Biol. 5: 109-118), in which the following species appear: (2) Lactobacillus casei subsp. rhamnosus CETC 276 (type strain); (3) Lb.casei subsp. rhamnosu CETC 278; Lb. CETC 748 plantarum (type strain); (5) Lb. CETC 573 halotolerans (type strain); (6) Lb. CETC 90 curvatus (type strain); (7) Lactobacillus sp .; (8) Lb. CETC 221 plantarum; (9) Lb. casei subsp. casei 102S; (10) Lb. casei subsp. case 63H; (11) Lb. casei subsp. casei 64H (lac-); (12) Lb. bavaricu CETC 980 (type strain); (13) Lb. casei subsp. casei CETC 4040 (14) Lb. fer entum CETC 4007 (type strain). The molecular step pattern is marked with the number 1.

FIGURE IB.- Same as figure 1A, with the following species: (2 Lactococcus lactis ML3 CNRZ 141; (3) Lb. casei subsp. Casei ATC 393 (type strain); (4) Lb. curvatus CETC 904; (5) Lb. plantaru CETC 748; Lactobacillus sake CETC 906. The molecular passage pattern is marked with the number 1.

FIGURE 2.- Bands amplified by the chain polymerization reaction performed on cells used by the rapid method described, of the following species: (2) Lactobacillus case subsp. rhamnosus CETC 276 (type strain); (3) Lb. casei subsp. case ATCC 393 (type strain); (4) Lb. bavaricus CETC 980 (type strain); (5 Lb. plantarum CETC 748 (type strain); (6) Lb. halotolerans CETC 57 (type strain); (7) Lb. fermentum CETC 4007 (type strain); (8) Lb sake CETC 906 (type strain); Lb. curvatus CETC 904 (type strain) Lb. delbruekii subsp. Bulgaricus CETC 4005 (type strain); (12) Lb brevis CETC 4121 (type strain) Line 1 as in figure 1.

FIGURE 3.- Bands amplified by the chain polymerization reaction performed on purified DNA of different strains from Lb. casei subsp. casei: (2) CETC 475 (ATCC 393); (3) ATCC 393; (4) 64H; (5) 64H (lac-); CETC 4040. Line 1, as in Figure 1.

FIGURE .- Radiographic film corresponding to the development and hybridization of a "dot blot" of three membranes containing dots with DNA from the type strains (column 1) Lb. casei subsp. casei, (column 2) Lb. plantarum, (column 3) Lb. curvatus y (column 4) Lb. sake, amplified by PCR with the oligonucleotide described in the text, and also non-amlified DNA. The membrane (A) was hybridized with a probe selected from Lb. curvatus; the membrane (B) was hybridized with a probe selected from Lb. sake; and the membrane (C) was hybridized with a probe selected from Lb. plantarum

Claims

1.- PROCEDURE TO OBTAIN SPECIFIC STRAP OR SPECIES PROBES, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel that come from the amplification by polymerases of the organism DNA for which nonspecific oligonucleotides have been used , and characterized in that said selected fragments of DNA are labeled and then used as they are in a hybridization in which the DNA of the samples has been fixed to a solid support.

2.- A procedure based on the extraction of the A contained in one or several bands visualized in an electrophoresis gel, which comes from the amplification by DNA of an organism for which nonspecific oligonucleotides have been used, and which is characterized by DNA of the samples have been obtained by any method.

3. - A simple and quick procedure to obtain specific strain of species or species, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which are derived from the amplification by DNA polymers of an organism for that nonspecific oligonucleotides have been used, and characterized in that said selected DNA fragments are labeled by methods that involve the use of radioactive isotopes, or by radioactive methods.

4. - A simple and fast procedure to obtain specific strain of species or species, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which come from the polymer amplification of the DNA of an organism for that nonspecific oligonucleotides have been used, and that is characterized by said fragments are labeled and then used as a probe in a hybridization whose astringency conditions will be controlled by varying the temperature, formamide concentration, salts in solution, and other factors.

5.- A simple and fast procedure to obtain specific probes of lineage or species, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which come from the polymerase amplification of the DNA of an organism to that nonspecific oligonucleotides have been used, and that is characterized in that the DNA of the samples is fixed to a solid support whether or not previously treated with restriction enzymes or polymerases.

6.- A simple and fast procedure to obtain specific probes of lineage or species, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which come from the polymerase amplification of the DNA of an organism to that nonspecific oligonucleotides have been used, and which is characterized in that after hybridization a detection method will be applied such that by itself, or as a consequence of a chemical or enzymatic reaction, it absorbs or emits electromagnetic radiation.

7. - A simple and quick procedure to obtain specific probes of lineage or species, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which come from the polymerase amplification of the DNA of an organism to that nonspecific oligonucleotides have been used, and that is characterized in that the DNA of the samples is fixed to a solid support such as membranes of nitrocellulose, nylon, plastics, glass, or any other.

8.- A simple and fast procedure to obtain specific strain or species probe, based on the extraction of DNA contained in one or several bands visualized in an electrophoresis gel, which come from the polymerase amplification of the DNA of an organism to that nonspecific oligonucleotides have been used, and which is characterized by that it can be applied to any prokaryotic and eukaryotic species.