CN101113471A - Method for detecting food-borne pathogenic Enterobacteriaceae using multiplex fluorescent PCR technology - Google Patents

Abstract

The invention discloses a method for detecting food-borne pathogenic Enterobacteriaceae using a composite fluorescence PCR technique, which belongs to the technical field of bacterial inspection, and the main technical solution is to design a primer set sequence. Pathogenic Enterobacteriaceae are common pathogenic bacteria in food, which seriously threaten people's health. Rapid and accurate detection of pathogenic Enterobacteriaceae in food is the main prerequisite for effective prevention and control of pathogenic bacterial infections. Foodborne pathogenic enterobacteria that need to be detected mainly include the following: Shigella, Salmonella, Yersinia enterocolitica, Escherichia coli and Escherichia coli O157:H7. For the above-mentioned target bacteria, the present invention overcomes the shortcomings in the prior art, and provides a rapid and low-cost method for detecting Shigella, Salmonella, Yersinia enterocolitica, Escherichia coli and Methods for Escherichia coli O157:H7. This method can use two tubes of PCR reactions, and can initially screen out the above-mentioned several pathogenic microorganisms.

Description

Method for detecting food-borne pathogenic Enterobacteriaceae using multiplex fluorescent PCR technology

technical field

The invention relates to a bacteria inspection technology, specifically a technology for detecting pathogenic bacteria, especially food-borne pathogenic enterobacteriaceae, by using fluorescent PCR reaction.

Background technique

Common pathogenic bacteria in food is one of the main causes of food poisoning, which can lead to a variety of diseases and seriously threaten people's health. The common pathogenic bacteria in food are mainly Enterobacteriaceae. Rapid and accurate detection of pathogenic bacteria in food is a prerequisite for effective prevention and control of pathogenic bacteria infection. With the development of molecular biology, the bacterial identification methods in food inspection and quarantine work have developed from the traditional simple biochemical experiment level to molecular biological methods such as PCR, probe hybridization and other technologies. In the short term, it is still difficult to replace traditional biochemical identification. Currently, molecular biology methods are mainly used for primary screening of a large number of samples. Foodborne Enterobacteriaceae that need to be detected mainly include the following: Shigella, Salmonella, Yersinia enterocolitica, Escherichia coli and Escherichia coli O157:H7, etc.

Contents of the invention

For the above-mentioned target bacteria, the present invention overcomes the shortcomings in the prior art, and provides a rapid and low-cost method for detecting Shigella, Salmonella, Yersinia enterocolitica, Escherichia coli and Escherichia coli O157:H7 method.

The method includes: applying the method to detect food-borne pathogenic microorganisms using a primer set and matching PCR reaction conditions. Wherein the whole sequences of primers and probes are as follows:

Primers:

Primer sequence one: 5'CCGTGTACGCTTAGTCGCTTAACCTC

Primer sequence two: 5'TCTTTAAGAATCTGGATCAAGCTGAAA

Primer sequence three: 5'CGAATGTGTCACCACATTCTCACCT

Primer sequence four: 5'GGTAAAGAGGTTCTGACTACACGATG

Primer sequence five: 5'CCCCATCGTGTAGTCAGAACCTCT

Primer sequence six: 5'ATTTGAAGAGGTTTTTAACTACATGTTAT

Primer sequence seven: 5'ATAACATGTAGTTAAAACCTCTTCAAAT

Primer sequence eight: 5'TGAACAGGAGGTTTCTGCGTTAG

Primer sequence nine: 5'ATATGTCAACCTCTGACTGATAGTCTGA

Primer sequence ten: 5'CTTTATGAAAGCCTGCGAGTAAAG

Primer sequence eleven: 5'CTGTTATGCTGGCTATCAGTCCTCT

The invention provides a more rapid and low-cost method for detecting food-borne pathogenic enterobacteriaceae by means of a fluorescent PCR method.

The present invention is achieved through the following technical solutions:

(1) Design specific oligonucleotide primers for the detection of fluorescent dye chimeric fluorescent PCR technology;

(2) The primers are integrated so that they do not interfere with each other.

(3) Amplify the sample template to be tested with the primer sequence set, and carry out specific amplification of the target gene;

(4) During the amplification process, use a fluorescent PCR instrument for real-time fluorescence light intensity measurement and measure the melting curve of the synthesized DNA fragment after the PCR reaction, and transmit the data to the computer for analysis through supporting software. Various The peak of the specific melting curve for pathogenic microorganisms.

System 1 of the present invention uses specific primer sets to amplify the genomes of Salmonella, Shigella, and Yersinia enterica, all of which produce fluorescent signals and generate corresponding melting curve peaks, and no false positives or false negatives are found. result.

In the present invention, the constituent ratios of each component in the fluorescent PCR reaction system are as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 2 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence three for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence four for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence five for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence six for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence 7 for fluorescent chimeric method: 10μmol/L 0.6μL

DNA sample 1μL

Double distilled water 8.5μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 60°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

Fluorescent PCR results Use a fluorescent PCR instrument for real-time fluorescence light intensity measurement during the amplification process, and perform gradient temperature rise to determine the melting temperature of the amplified fragment after the PCR amplification program ends. The fluorescence generated by specific amplification can be observed by transferring the data to the computer for analysis through supporting software, and the peak value of the melting curve (77°C±0.3°C) specific to the specific fragment of Shigella can be obtained: small intestine Melting curve peak (74°C ± 0.3°C) specific to the Yersinia specific fragment Melting curve double peak (81°C ± 0.3°C and 84.5°C ± 0.3°C) specific to the Salmonella spp. specific fragment.

If the specific melting curve peak (77°C ± 0.3°C) is obtained for the specific fragment of Shigella, it proves that Shigella exists in the sample to be tested; if the specificity for Yersinia enterica is obtained The peak value of the melting curve (74°C±0.3°C) unique to the fragment proves that the sample to be tested contains Yersinia enterica; the double peak of the melting curve (81°C±0.3°C and 84.5 °C ± 0.3 °C), it proves that the sample to be tested contains Salmonella. If there are other results, it means that the failure of this test cannot determine the presence of Shigella, Salmonella and Yersinia enterica, and a new test is required.

System 2 of the present invention uses a specific primer set to amplify the genomes of Escherichia coli O ₁₅₇ :H ₇ strain and hemorrhagic Escherichia coli to generate fluorescent signals and generate corresponding melting curve peaks, and no false positives or false negatives were found. result.

In the present invention, the constituent ratios of each component in the fluorescent PCR reaction system are as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 8 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 9 for fluorescent chimeric method: 10μmol/L 0.3μL

The sequence of primers used in the fluorescent chimeric method ten: 10μmol/L 0.3μL

Primer sequence 11 for fluorescent chimeric method: 10μmol/L 0.3μL

DNA sample 1μL

Double distilled water 10.3μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 60°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

Fluorescent PCR results Use a fluorescent PCR instrument for real-time fluorescence light intensity measurement during the amplification process, and perform gradient temperature rise to determine the melting temperature of the amplified fragment after the PCR amplification program ends. Transfer the data to the computer and analyze it through the supporting software to observe the fluorescence generated by specific amplification, and obtain the peak melting curve (77.5°C±0.3°C) specific to the specific fragment of Escherichia coli O ₁₅₇ :H ₇ strain and the peak of the melting curve (74°C ± 0.3°C) specific to the specific fragment of Escherichia coli.

If the specific melting curve peak (77.5°C ± 0.3°C) is obtained for the specific fragment of Escherichia coli O ₁₅₇ :H ₇ strain, it proves that there is Escherichia coli O ₁₅₇ in the sample to be tested: H ₇ strain; The peak value of the melting curve (74°C±0.3°C) specific to the specific fragment of Escherichia coli proves that the sample to be tested contains hemorrhagic Escherichia coli. If other results appear, it indicates that the failure of this test cannot determine the presence of Escherichia coli O ₁₅₇ : H ₇ strain and hemorrhagic Escherichia coli.

Compared with the prior art, the beneficial effects of the present invention are: the identification method based on fluorescent PCR is suitable for directly amplifying target genes from clinical samples such as patient's bacteria-containing body fluid, food and body fluid culture, thereby bacteria, and using this method Shigella, Salmonella, Yersinia enterica, Escherichia coli O ₁₅₇ : H ₇ strain and hemorrhagic Escherichia coli can be detected by only one two-tube fluorescent PCR reaction, which can save detection cost and time. The fluorescent PCR method has the characteristics of accurate detection, strong specificity and high sensitivity, and can quickly and accurately identify specific target bacteria. It uses PCR method to amplify bacterial target genes, avoids repeated cultivation and saves time; PCR identification method is not affected by culture conditions and physiological state of bacteria, and is more accurate than physiological and biochemical identification methods.

Description of drawings

Fig. 1 The fluorescence signal intensity of SYBR Green in a fresh meat sample to detect the DNA of the sample to be tested by the multiplex fluorescent PCR technique.

Figure 2 The peak melting temperature of the amplified product detected by multiplex fluorescent PCR technology in a fresh meat sample to be tested.

Figure 3: The fluorescence signal intensity of SYBR Green in a biscuit sample to be tested by multiplex fluorescent PCR technology to detect the DNA of the sample to be tested.

Fig. 4 The peak melting temperature of the amplified product detected by multiplex fluorescent PCR technology in a biscuit sample to be tested.

Figure 5. The composite fluorescent PCR technology in a raw milk sample to be tested detects the DNA of the sample to be tested, and the fluorescence signal intensity of SYBR Green.

Fig. 6 The peak melting temperature of the amplified product detected by multiplex fluorescent PCR technology in a raw milk sample to be tested.

Figure 7: In a canned beef sample to be tested, the DNA of the sample to be tested was detected by the multiplex fluorescent PCR technique, and the fluorescence signal intensity of SYBR Green.

Fig. 8 The peak melting temperature of the amplified product detected by multiplex fluorescent PCR technology in a canned beef sample to be tested.

Figure 9: In a sample of dried tofu to be tested, the composite fluorescent PCR technology detects the DNA of the sample to be tested, and the fluorescence signal intensity of SYBR Green.

Figure 10 shows the peak melting temperature of the amplified product detected by multiplex fluorescent PCR technology in a dried tofu sample to be tested.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example 1

Sample: An export of fresh meat.

Use routine physiological and biochemical methods to detect suspected Shigella colonies, and then perform the following composite fluorescent PCR technology to detect foodborne pathogenic bacteria:

1. Sample Processing

(1) Take 100 grams of the sample to be tested and pulverize it.

(2) Dissolve in 1 liter of nutrient broth and incubate at 37°C for 8 hours.

2. DNA extraction

Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 2 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence three for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence four for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence five for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence six for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence 7 for fluorescent chimeric method: 10μmol/L 0.6μL

DNA sample 1μL

Double distilled water 8.5μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 65°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

A total of 2 tubes of PCR experiments were carried out, in which the DNA samples were added: the DNA of the sample to be tested; the negative control without samples.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the sample to be tested produced obvious SYBR Green fluorescence, and the results are shown in Figure 1. The negative control did not produce SYBR Green fluorescence results during the PCR process. By observing the fluorescence curve of the melting temperature of the amplified product, it can be found that the characteristic peak of Shigella is shown in Figure 2.

The SYBR Green fluorescence intensity signal represented by the curve in Figure 1 is the result of DNA amplification in the sample.

The melting temperature peak represented by the curve in Figure 2 is a characteristic peak of Shigella in the sample.

Experiments showed that the samples contained Shigella bacteria.

Three days later, routine microbial culture and biochemical testing proved that the target colony was Shigella flexneri, and one of the strains was named CIQ810. The fluorescent PCR test results were consistent with the biochemical test results.

Example 2

Sample: Somewhere exports cookies.

Use routine physiological and biochemical methods to detect suspected colonies of Salmonella, and then perform the following composite fluorescent PCR technology to detect foodborne pathogens:

1. Sample Processing

(1) Take 100 grams of the sample to be tested and pulverize it.

(2) Dissolve in 1 liter of nutrient broth and incubate at 37°C for 8 hours.

2. DNA extraction

Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 2 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence three for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence four for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence five for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence six for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence 7 for fluorescent chimeric method: 10μmol/L 0.6μL

DNA sample 1μL

Double distilled water 8.5μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 65°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

A total of 2 tubes of PCR experiments were carried out, in which the DNA samples were added: the DNA of the sample to be tested; the negative control without samples.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the sample to be tested produced obvious SYBR Green fluorescence, and the results are shown in Figure 3. The negative control did not produce SYBR Green fluorescence results during the PCR process. By observing the fluorescence curve of the melting temperature of the amplified product, it can be found that the characteristic peak of Salmonella is shown in Figure 4.

The SYBR Green fluorescence intensity signal represented by the curve in Figure 3 is the result of DNA amplification in the sample.

The melting temperature peak represented by the curve in Fig. 4 is a characteristic peak of Salmonella in the sample.

Experiments showed that the samples contained Salmonella.

Three days later, routine microbial culture and biochemical testing proved that the target colonies tested were Salmonella enteritidis, and one of the strains was named CIQ815. The fluorescent PCR test results were consistent with the biochemical test results.

Example 3

Sample: Raw milk sent for inspection somewhere.

Use conventional physiological and biochemical methods to detect suspected Yersinia enterica colonies, and then perform the following composite fluorescent PCR technology to detect foodborne pathogenic bacteria:

1. Sample Processing

(1) Take 100 grams and centrifuge.

(2) The precipitate was redissolved in 1 liter of nutrient broth and incubated at 37°C for 8 hours.

2. DNA extraction

Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 1 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 2 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence three for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence four for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence five for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence six for fluorescent chimeric method: 10μmol/L 0.6μL

Primer sequence 7 for fluorescent chimeric method: 10μmol/L 0.6μL

DNA sample 1μL

Double distilled water 8.5μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 65°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

A total of 2 tubes of PCR experiments were carried out, in which the DNA samples were added: the DNA of the sample to be tested; the negative control without samples.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the sample to be tested produced obvious SYBR Green fluorescence, and the results are shown in Figure 5. The negative control did not produce SYBR Green fluorescence results during the PCR process. By observing the fluorescence curve of the melting temperature of the amplified product, it can be found that the characteristic peak of Yersinia enterica is shown in Figure 6.

The SYBR Green fluorescence intensity signal represented by the curve in Figure 5 is the result of DNA amplification in the sample.

The melting temperature peak represented by the curve in Fig. 6 is a characteristic peak of Yersinia enterica in the sample.

Experiments showed that the samples contained Yersinia enterica.

Three days later, routine microbial culture and biochemical testing proved that the target colony was Yersinia enterica, and one of the strains was named CIQ919. The fluorescent PCR test results were consistent with the biochemical test results.

Example 4

Sample: Canned meat sent for inspection somewhere.

Detect Escherichia coli O ₁₅₇ :H ₇ suspected colonies with routine physiological and biochemical methods, and then carry out the following composite fluorescent PCR technology to detect the detection of food-borne pathogens:

1. Sample Processing

(1) Take 100 grams of the sample to be tested and pulverize it.

(2) The treated samples were dissolved in 1 liter of nutrient broth and incubated at 37° C. for 8 hours.

2. DNA extraction

Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 8 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 9 for fluorescent chimeric method: 10μmol/L 0.3μL

The sequence of primers used in the fluorescent chimeric method ten: 10μmol/L 0.3μL

Primer sequence 11 for fluorescent chimeric method: 10μmol/L 0.3μL

DNA sample 1μL

Double distilled water 10.3μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 60°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

A total of 2 tubes of PCR experiments were carried out, in which the DNA samples were added: the DNA of the sample to be tested; the negative control without samples.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the sample to be tested produces obvious SYBR Green fluorescence, and the results are shown in Figure 7. The negative control did not produce SYBR Green fluorescence results during the PCR process. By observing the fluorescence curve of the melting temperature of the amplified product, it can be found that the characteristic peak of E. coli O ₁₅₇ :H ₇ is shown in Figure 8.

The SYBR Green fluorescence intensity signal represented by the curve in Figure 7 is the result of DNA amplification in the sample.

The melting temperature peak represented by the curve in Fig. 8 is a characteristic peak of Escherichia coli O ₁₅₇ : H ₇ in the sample.

Experiments showed that the sample contained Escherichia coli O ₁₅₇ :H ₇ .

Three days later, routine microbial culture and biochemical testing proved that the target colony was Escherichia coli O ₁₅₇ :H ₇ , and one of the strains was named CIQ869. The fluorescent PCR test results were consistent with the biochemical test results.

Example 5

Sample: Dried tofu sent for inspection somewhere.

Use conventional physiological and biochemical methods to detect suspected colonies of hemorrhagic Escherichia coli, and then perform the following composite fluorescent PCR technology to detect foodborne pathogens:

1. Sample Processing

(1) Take 100 grams of the sample to be tested and crush it.

(2) The treated samples were dissolved in 1 liter of nutrient broth and incubated at 37° C. for 8 hours.

2. DNA extraction

Take 1 mL of nutrient broth and let it stand in an ice bath for 5 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 100 μL of lysozyme solution, incubate at 37°C for 10 minutes, and add 500 μL of TE buffer , shake to mix. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 12,000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (2mol/L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant , washed once with 70% cold ethanol, centrifuged at 12,000 rpm for 5 minutes at room temperature, discarded the supernatant, added 50 μL TE solution, and stored at -20°C.

3.PCR amplification

The composition ratio of each component in the PCR reaction system is as follows:

Composition Concentration Sample Amount

PCR system premix 2 times 12.5μL

Primer sequence 8 for fluorescent chimeric method: 10μmol/L 0.3μL

Primer sequence 9 for fluorescent chimeric method: 10μmol/L 0.3μL

The sequence of primers used in the fluorescent chimeric method ten: 10μmol/L 0.3μL

Primer sequence 11 for fluorescent chimeric method: 10μmol/L 0.3μL

DNA sample 1μL

Double distilled water 10.3μL

Total volume 25μL

The fluorescent PCR amplification program and the melting temperature program for determining the amplified fragment are:

(1) 10 minutes at 95°C;

(2) 95°C for 15 seconds;

(3) 30 seconds at 60°C;

(4) Go back to step (2) and repeat 40 times;

(5) 95°C for 2 minutes;

(6) Gradient temperature increase from 60°C to 95°C by 0.2°C per second.

A total of 2 tubes of PCR experiments were carried out, in which the DNA samples were added: the DNA of the sample to be tested; the negative control without samples.

4. Observation of the fluorescence PCR pattern of the tested sample

During the fluorescent PCR process, it can be observed that the sample to be tested produces obvious SYBR Green fluorescence, and the results are shown in Figure 9. The negative control did not produce SYBR Green fluorescence results during the PCR process. By observing the fluorescence curve of the melting temperature of the amplified product, it can be found that the characteristic peak of hemorrhagic E. coli is shown in Figure 10.

The SYBR Green fluorescence intensity signal represented by the curve in Figure 9 is the result of DNA amplification in the sample.

The melting temperature peak represented by the curve in Fig. 10 is a characteristic peak of hemorrhagic Escherichia coli in the sample.

Experiments showed that the sample contained hemorrhagic E. coli.

Three days later, routine microbial culture and biochemical testing proved that the target colony was Escherichia coli, and one of the strains was named CIQ639. The fluorescent PCR test results were consistent with the biochemical test results.

sequence list

SEQUENCE LISTING

<110> Tianjin Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China

<120> Method for detecting food-borne pathogenic Enterobacteriaceae using multiplex fluorescent PCR technology

<130>2007118

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Claims (5)

1. a method of using composite fluorescence PCR technology for detection food-borne pathogenic enterobacteria is characterized in that, employed primer sets sequence is as follows:

Primer:

One: 5 ' CCGTGTACGCTTAGTCGCTTAACCTC of primer sequence

Two: 5 ' TCTTTAAGAATCTGGATCAAGCTGAAA of primer sequence

Three: 5 ' CGAATGTGTCACCACATTCTCACCT of primer sequence

Four: 5 ' GGTAAAGAGGTTCTGACTACACGATG of primer sequence

Five: 5 ' CCCCATCGTGTAGTCAGAACCTCT of primer sequence

Six: 5 ' ATTTGAAGAGGTTTTAACTACATGTTAT of primer sequence

Seven: 5 ' ATAACATGTAGTTAAAACCTCTTCAAAT of primer sequence

Eight: 5 ' TGAACAGGAGGTTTCTGCGTTAG of primer sequence

Nine: 5 ' ATATGTCAACCTCTGACTGATAGTCTGA of primer sequence

Ten: 5 ' CTTTATGAAAGCCTGCGAGTAAAG of primer sequence

Primer sequence 11: 5 ' CTGTTATGCTGGCTATCAGTCCTCT.

2. a kind of method of using composite fluorescence PCR technology for detection food-borne pathogenic enterobacteria according to claim 1 is characterized in that each component composition is as follows in the PCR reaction system 1:

Constituent concentration application of sample amount

2 times of 12.5 μ L of PCR system premixture

One: 10 μ mol/L 0.3 μ L of primer sequence

Two: 10 μ mol/L 0.3 μ L of primer sequence

Three: 10 μ mol/L 0.6 μ L of primer sequence

Four: 10 μ mol/L 0.3 μ L of primer sequence

Five: 10 μ mol/L 0.3 μ L of primer sequence

Six: 10 μ mol/L 0.6 μ L of primer sequence

Seven: 10 μ mol/L 0.6 μ L of primer sequence

DNA sample 1 μ L

Distilled water 8.5 μ L

Cumulative volume 25 μ L.

3. a kind of method of using composite fluorescence PCR technology for detection food-borne pathogenic enterobacteria according to claim 1 is characterized in that each component composition is as follows in the PCR reaction system 2:

Constituent concentration application of sample amount

2 times of 12.5 μ L of PCR system premixture

Eight: 10 μ mol/L 0.3 μ L of primer sequence

Nine: 10 μ mol/L 0.3 μ L of primer sequence

Ten: 10 μ mol/L 0.3 μ L of primer sequence

Primer sequence 11: 10 μ mol/L, 0.3 μ L

DNA sample 1 μ L

Distilled water 10.3 μ L

Cumulative volume 25 μ L.

4. a kind of method of using composite fluorescence PCR technology for detection food-borne pathogenic enterobacteria according to claim 1 is characterized in that, the melting temperature (Tm) program of fluorescent PCR amplification program and mensuration amplified fragments is:

(1) 95 ℃ 10 minutes;

(2) 95 ℃ 15 seconds;

(3) 65 ℃ 30 seconds;

(4) got back to for (2) step, repeat 40 times;

(5) 95 ℃ 2 minutes;

(6) 60 ℃ to 95 ℃ per seconds of gradient increased temperature rise 0.2 ℃.

5. the described a kind of application of composite fluorescence PCR technology aspect detection food-borne pathogenic enterobacteria of using of claim 1.

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