LT3828B - Process for detecting salmonella bacteries and probe for the same - Google Patents

Abstract

Invention creates a new determination method of Salmonella bacterium. The method is distinguished by the tested bacterium being used in the PCR applying probes 5'-GTAAATCCGGTTCACTTTAACAC-3' and 5'-GAGTAAATCACTTCACCTACGTG-3', complementary for the gene S.typhimurium 23S rRNR, products of amplification are analysed by electrophoresis and decision of the specific character of determination is taken according to the restriction analysis of amplification products, using restriction endonucleus Eco 130 l. It was empirically established that oleonucleotides probes are general and are specific only to the genus of Salmonella bacterium. Detection sensitivity limit - 50 bacterium of 10 μl sample.

Description

The invention defines a novel method for the detection of Salmonella bacteria and oligonucleotide probes and can be used in medicine, the food industry, and veterinary medicine.

The oldest method for testing Salmonella bacteria used in microbiological laboratories, including culturing the bacteria in selective media and characterizing the morphological and biochemical characteristics of the resulting colonies, lasts on average 4 days. This method is also used in Lithuanian microbiological laboratories (MeTOnnuecKne yKa.3a.HHM no MHKpoŪHOJiornnecKOH gnai'HOCTHKe 3a6ojieBaHHH, Bbi3BaHHbix 3HTepo6aKTepnaMH, MocKBa, 1984).

The use of nucleic acid hybridization technology is a qualitative leap in the diagnosis of pathogenic microorganisms. This methodology is based on the property that the unique polynucleotide sequence of the microorganism being tested hybridizes to a specifically labeled nucleic acid probe. A negative property of these assays is lack of sensitivity (WO 89/05359, C12Q 1/68, 1989; EP 0383509, C12Q 1/68, 1990; WO 90/11370, C12Q 1/68, 1990).

Closest to the proposed technical solution is a patent (WO 90/11370, C12Q 1/68, 1990), which uses 32p-labeled DNA probes complementary to the Omp A gene of Salmonella typhimurium to detect Salmonella bacteria. The bacteria to be tested are grown to OD = 0.7, 100 µl of the bacterial suspension is filtered through a nitrocellulose filter and the DNA is immobilized by irradiation of the filters for 4 minutes with a UV lamp. Filters are prehybridized for 45 minutes and, after the addition of the 32p-labeled probe, are hybridized for an additional 1.5 hours. Filters are cleaned and radioautographed for 3 days.

Lack of sensitivity - insufficient sensitivity, long duration.

Recently, a polymerase cyclic reaction method has been introduced in molecular biology (Saiki R.K. et al., Science 239, 487-491 (1988)). It is a method to obtain microgram quantities of desired DNA from a very small amount of DNA matrix but has not yet been directly used for Salmonella detection.

The object of the present invention is to provide a method for rapid and specific detection of Salmonella bacteria. This objective is achieved by the application of a polymerase cyclic reaction (PCR) to the detection of Salmonella bacteria, i. using oligonucleotide DNA probes complementary to the Salmonella typhimurium 23S rRNA gene and thermostable DNA polymerase, alternating temperature cycles from denaturation to polymerization temperatures, enzymatically reproduced from the used DNA template by Salmonella-specific DNA amplification, no amplification of various non-Salmonella bacteria DNA.

The oligonucleotide probes of the present invention have the following characteristics:

1. Follow:

> -GTAAATCCGGTTCACTTTAACAC-3 '

5'-GAGTAAATCACTTCACCTACGTG- 3 ·

The probes were synthesized by the Cyanoethyl Phosphoramidite method in the Nucleic Acid Chemistry Sector of the Institute of Biotechnology.

2. It has been empirically determined that these probes are universal for bacteria of the genus Salmonella, i.e. The use of probes in the polymerase cyclic reaction allows the detection of 13 Salmonella species in the sample (Table 1):

S.brandenburg, S.choleraesuis, S.enteritidis, S.haifa, S.infantis, S.isangi, Sj ava, S.newport, S.ngor, S.Stanley, S.thompson, S.typhimurium, S.tshionqwe .

3. It has been empirically determined that the probes are specific to bacteria of the genus Salmonella, ie, using probes containing Acinetobacter, Citrobacter, Citrobacter freundii, Escherichia coli, Enterobacter, Enterobacter aerogenes, Enterobacter cloacae, Hafnia, Yersinia enterocolitica, Klebsiella, Proteus, The polymerase cyclic reaction of Providencia rettgeri, Pseudomonas, Pseudomonas aeruginosa, Shigella flexneri, Shigella sonnei, Serratia does not occur (Table 2).

FIG. Electrophoretic PCR products obtained with various

S.typhimurium in bacterial counts, analysis. Quantities of bacteria used for PCR; 1-5x10 ⁶ , 2-5x10 ⁵ , 3-5x10 ⁴ , 4-5x1 () 3, 5-5xlo2, 6-5x10, 7-5 in the bacterial sample,

Fragment sizes of 8-pBR-Alu I fragments in base pairs.

FIG. Electrophoretic analysis of PCR products after incubation with Ecol30 I. 9 - S.typhimurium PCR product, 10 - S.typhimurium PCR product-Ecol30 I, 11S.enteritidis PCR product -Ecol30 I, 12 - S.typhimurium PCR product-Eco64 I, Fragment sizes of 13-pBR-Alu I fragments in base pairs.

3. Electrophoretic analysis of PCR products obtained with various bacteria. Fragment sizes of 14,28-pBR-Alu I fragments in base pairs. Bacteria used for PCR: 15, 16, 17 - S.enteritidis; 18 - S.tshionqwe;

19,20,21- S.infantis; 22.23 - S.brandenburg; 24,25,27S.typhimurium; 26-Y.enterocolitica.

The invention is illustrated by the following examples.

An example. Evaluation of the susceptibility of the polymerase cyclic reaction used to detect Salmonella bacteria.

Bacterial strains.

All bacterial strains (Tables 1,2) used in this work were obtained at the Microbiological Laboratory of Vilnius Hygiene Center.

Polymerase cyclic reaction (PCR).

The reaction is performed in 100 μΐ of a mixture of 67 mM Tris-HCl (pH 8.4), 2 mM MgCl ₂ , 16.6 mM (NH 4 SO, 10 mM mercaptoethanol, 1.25 mM dATP, 1.25 mM dTTP, , 25 mM dGTP, 1.25 mM dCTP, 0.001 mM primer, 20 µg bovine serum albumin, 2 units Taq polymerase The bacteria to be tested were grown overnight in 4 ml of LB medium at 37 ° C. 100 μΐ cell suspensions were centrifuged in a microcentrifuge at 11000 rpm. For 5 minutes, the pellet is suspended in 100 μΐ of sterile H ₂ O and boiled for 10 minutes at 100 ° C. 10 μΐ of this suspension is used in the polymerase cyclic reaction, empirically selected conditions for the polymerase cyclic reaction, 25 cycles each : 95 ° C for 1 min, 58 ° C for 1 min, 70 ° C for 1 min After completion of the PCR reaction, the mixture is extracted twice with chloroform and 10 μΐ of the aqueous phase is analyzed electrophoretically on a 1.5% agarose gel. fragment of base pore size s (Fig. 1).

For PCR sensitivity evaluation, S.typhimurium culture was grown overnight in 4 ml LB medium, titrated in saline and plated at 100 μΐ on agarized LB medium to calculate viable cell titer. PCR is performed on various bacterial levels. From the results presented in Fig. 1, we can see that the sensitivity limit of the test is 50 samples of Salmonella bacteria in 10 μΐ.

An example. Investigation of oligonucleotide probes for use in PCR.

restriction enzymes

Amplified product identification.

Amplification products were identified empirically from many of those used in Ecol30 I. Salmonella-PCR product - 10 μ - was incubated for 1 h. At 37 ° C, 20 μΐ of the reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2> 100 mM NaCl, and restriction endonuclease Ecol30 I. The restriction products were analyzed electrophoretically on a 2% agarose gel. Fig. 2 illustrates S.typhimurium- and

Electrophoretic image of S.enteritidis-PCR products restricted to Ecol30 I. Absolutely identical images in Fig. 2 are obtained after PCR products using S.brandenburg, S.choleraesuis, for PCR reaction.

S.haifa, S, infantis, S.isangi, S.java, S, newport,

S.ngor, S.Stanley, S.thompson, S.tshiongwe bacteria, incubation with Ecol30 I.

Probes 5'-GTAAATCCGGTTCACTTTAACAC-3 'and 5 --GAGTAAATCACTTCACCTACGTG-3>, complementary to the Salmonella typhimurium 23S rRNA gene, were used to detect Salmonella bacteria. Samples 1 and 2 were tested for a range of bacteria of the genus Salmonella and other genera belonging to the Enterobacteriaceae family.

table

PCR results with Salmonella bacteria

For example, No. The title Investigate strains number The fragment ampli- fixes (+) or not (-) 3. S. brandenburcf 7th + 4. S.choleraesuis 1 + 5. S.enteretidis 42 + 6th S.haifa 1 + 7th S.infantis 6th + 8th S. isancji 2 + 9th S.ή open 1 + 10th S .newport 1 + 11th S.nqor 1 + 12th S. Stanley 1 + 13th S.thomoson 1 + 14th S.typhimurium 9th h 15th S.tshionqwe 1 +

table

PCR results with various non-Salmonella bacteria

For example, No. The title Investigate strains number The fragment ampli- fixes (+) or not (-) 16th Acinetobacter 1 - 17th Citrobacter 1 - 18th Citrobacter freundii 2 - 19th Escherichia coli 6th - 20th Enterobacter 4 - 21st Enterobacter aeroqenes 1 - 22nd Enterobacter cloacae 1 - 23rd Hafnia 5 - 24th Yersinia enterocolitica 1 - 25th Klebsiella 4 - 26th Proteus 1 - 27th Proteus vulqaris 1 - 28th Providencia rettqeri 1 - 29th Pseudomonas 2 - 30th Pseudomonas aeruqinosa 3 - 31st Shiqella flexneri 2 - 32. Shiqella bull 11th - 33. Serratia 1

3, the results in Tables 1.2 confirm that the use of oligonucleotide primers complementary to the Salmonella typhimurium 23S rRNA gene results in a very specific polymerase cyclic reaction, i.e. amplification occurs only from Salmonella bacterial DNA whereas no amplification from various other bacterial DNAs. Apparently, the probes used are versatile enough for bacteria of the genus Salmonella, since their use enables the detection of 13 different Salmonella species.

Advantages of the present invention:

1. Hypersensitivity of Salmonella detection, i.e. 50 bacteria in the sample are sufficient for the polymerase cyclic reaction.

2. The detection time of Salmonella bacteria is reduced to one day.

3. A restriction is used to identify the amplified product. The restriction enzyme Ecol30 I was empirically selected (Fig.2).

Claims (4)

DEFINITION OF INVENTION 1. Oligonucleotide probes - primers complementary to the Salmonella typhimurium 23S rRNA gene, having the sequence: 5′-GAG-TAA-ATC-ACT-TCA-CCT-ACG-TG-3 ′ and 5 ′ -GTA-AAT * CCG-GTT-CAC-TTT-AAC-AC-3 ′ for detection of Salmonella bacteria. 2. A method for detecting Salmonella bacteria, wherein the bacteria are tested by probes, characterized in that the bacteria are used in the polymerase cyclic reaction using the probes according to claim 1 electrophoretically and the detection is determined by analysis of the amplification products. analyzes the specificity of the restriction 3. The process of claim 2, wherein the polymerase has at least 25 cycles of cyclic reaction, each lasting 1 minute. 95 ° ± 1 ° C for 1 min. - 58 ° ± 1 ° C for 1 min. 70 ° ± 1 ° C. 4. The method of claim 2, wherein the amplification products are identified using the restriction endonuclease Ecol30 I.