CN1796571A - Method of multiplex fluorescence PCR - improved molecule beacon for detecting pathogenesis bacterium stemmed from eating source - Google Patents

Abstract

This invention relates to a method of detection of food-borne pathogenic bacteria using multiple fluorescent PCR and modified molecular beacons. Several pairs of primers and modified molecular beacon probes are designed according to the specific gene sequence of the pathogenic bacteria to be tested, which is amplified using fluorescent PCR and modified molecular beacon. The amplified product is crossbred with molecular beacon, and the fluorescent intensity of the reaction system is tested with the necessary circle times for intended threshold as a judgment for results, that is, negative for Ct of 0 or 40 and positive for Ct less than 38. This invention, which is advanced in high sensibility, significant specificity, simple operation and intuitionistic observation, is applicable in simultaneous detection of any two kinds, three kinds and four kinds of randomly combined bacteria and food-borne pathogenic bacteria as well as specimen in large amounts.

Description

Multiplex fluorescent PCR-improved molecular beacon method for detecting foodborne pathogenic bacteria

technical field

The invention relates to a method for detecting food-borne pathogenic bacteria with a fluorescent PCR molecular beacon.

Background technique

There is a high incidence of foodborne diseases such as Vibrio cholerae causing cholera, Salmonella typhi and Shigella causing typhoid and dysentery respectively. Cholera, typhoid, and dysentery are key infectious diseases in my country. The incidence of food poisoning caused by Vibrio parahaemolyticus, Salmonella, Staphylococcus aureus, Bacillus cereus, Proteus, etc. accounts for a relatively high proportion of the incidence of foodborne diseases in my country. At present, these food-borne pathogenic bacteria are still detected by the traditional methods of bacterial isolation, culture and identification. This traditional detection method is cumbersome to operate, time-consuming and labor-intensive. It usually takes 5 days, and the longest time is one month. Moreover, the biochemical and serological tests are unstable and the detection sensitivity is low.

With the development of molecular biology technology, people use PCR technology for the diagnosis of bacteria. For example, the patent application with the Chinese Patent Publication No. CN1526825 discloses a method for detecting Vibrio parahaemolyticus by utilizing the specificity of the pR72H sequence of Vibrio parahaemolyticus through modern molecular biological means such as DNA extraction, PCR amplification, and electrophoresis observation. However, the PCR technique is easy to contaminate, resulting in detection failure, and electrophoresis is required. Since the US PE company proposed the real-time PCR detection principle in 1995, real-time PCR has been widely used for its outstanding advantages of rapidity, quantification, no need for post-electrophoresis, and no cross-contamination.

Molecular beacon is a kind of molecular probe with neck ring configuration proposed by Tyagi et al. in 1996. It is designed based on the principle of nucleic acid base pairing and fluorescence resonance energy phenomenon. During the annealing stage of PCR amplification, molecular beacon and The generated target sequence binds to emit fluorescence, and in the extension stage, it breaks away from the target sequence without interfering with amplification. As the number of cycles increases, the amount of molecular beacons bound to the template also increases, and the final fluorescence intensity is proportional to the amount of template.

However, the real-time PCR method currently used is a single fluorescent PCR method, which can only detect one pathogenic bacteria, and the detection speed and sensitivity still cannot meet the requirements. With the change of the ecological environment and the abuse of antibiotics, the clinical symptoms caused by many pathogenic bacteria are becoming less and less typical, and it is often necessary to detect multiple bacteria at the same time to determine the pathogen. This puts forward higher requirements for rapid diagnostic technology: it can detect multiple pathogenic microorganisms quickly and at the same time. Therefore, it is urgent to develop a method for rapid diagnosis of multiple bacteria at the same time.

Contents of the invention

In order to overcome the shortcomings of the existing technology, such as cumbersome operation and long time consumption, and the inability to quickly detect multiple food-borne pathogens at the same time, a method for detecting food-borne pathogens by multiple fluorescent PCR-improved molecular beacons is provided .

In order to solve the above-mentioned technical problems, the technical solution of the present invention is a method for detecting food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons, which comprises the following steps:

(1) Design multiple pairs of primers and multiple improved molecular beacon probes according to the specific gene sequences of various pathogenic bacteria to be tested;

(2) Prepare a reaction system for fluorescent PCR-improved molecular beacon amplification gene sequence;

(3) using the various fluorescent PCR-improved molecular beacons of step (1) to simultaneously amplify the sequence of multiple pathogenic bacteria specific genes to be tested;

(4) Using the hybridization between the molecular beacon and the amplification product to detect the fluorescence intensity of the reaction system, the Ct value of the number of cycles required to reach the set threshold is used as the result judgment standard, and the Ct value is 0 or 40: negative; Ct less than 38: Positive.

The reaction system of fluorescent PCR-improved molecular beacon amplification gene sequence is:

1×PCR buffer

MgCl ₂ 0.5～5mmol

dNTP each 0.05～1.5mmol

Each pair of primers 0.1～1.0μmol+0.1～1.0μmol

Each probe 0.1～1.0μmol

Taq enzyme 0.2～10U

Template 1～20μL

Total volume 20～100μL

The reaction conditions for the fluorescent PCR-improved molecular beacon amplification specific gene sequence are pre-denaturation at 90-100°C for 3-10 minutes, denaturation at 92-95°C for 15-60 seconds in 40-50 cycles, and annealing at 50-60°C 15-60 seconds, 70-72°C extension for 15-120 seconds.

The pathogenic bacteria are selected from Vibrio cholerae, Vibrio parahaemolyticus, Staphylococcus aureus, Listeria monocytogenes, Salmonella, Shigella, and O157: H7 Escherichia coli one or more combination.

The specific gene sequences of the pathogenic bacteria are Vibrio cholerae enterotoxin gene ctxA sequence, Vibrio parahaemolyticus thermostable direct hemolysin gene TDH sequence, Staphylococcus aureus thermostable nuclease gene NUC, Listeria monocytogenes Bacteria hemolysin gene hly sequence, Salmonella invasion gene ivnA sequence, Shigella invasion plasmid antigen H gene ipaH sequence, and O157: H7 E. coli hemolysin gene rfbE sequence.

The primers and probes of the improved molecular beacon are respectively:

A pair of primers for ctxA gene:

ctxA-F: 5′-TCCGGAGCATAGAGCTTGGA-3′,

ctxA-R: 5′-TCGATGATCTTGGAGCATTCC-3′

Probes for the ctxA gene:

HEX-5'- CCGTGGATTCATCATGCACCGCC ACGG-3'Dabcyl,

A pair of primers for TDH gene:

TDH-F: 5'-AAACATCTGCTTTTGAGCTTCCA-3',

TDH-R: 5′-CTCGAACAACAAACAATATCTCATCATCAG-3′,

Probes for the TDH gene:

FAM5′- CCGGGGTGTCCCTTTTTCCTGCCCCCGG -Dabcyl,

A pair of primers for NUC gene:

NUC-F: 5'-GGCAATACGCAAAGAGGTT-3'

NUC-L: 5'-CTTCTTCTATTTACGCCGTTATC-3'

Probes for NUC genes:

ROX-5'-CGATGCA GTCTAAGTAGCTCAGCAAATGCATC G-3'-DABCYL

A pair of primers for the hly gene:

hly-F: 5'-TGCAAGTCCTAAGACGCCA-3'

hly-L: 5'-CACTGCATCTCCGTGGTATACTAA-3'

Probes for the hly gene:

FAM-5'-CGCG CTTGTATATACTTATCGATTTCATCCGCGCG -3'-DABCYL

A pair of primers for the invA gene:

invA-F: 5'-GCGGAATATC(I)ATGACGCAGCT-3'

invA-L: 5'-CGCTACGTTTTGCTTCACGG-3'

Probes for the invA gene:

5'-FAM-CCG CCATTTGTATTGGTTGTTACGGC GG-DABCYL-3'

A pair of primers for ipaH gene:

ipaH-F: 5'-TGAAGGAAATGCGTTTCTATG-3'

ipaH-L: 5'-AGGGAGAACCAGTCCGTAAA-3'

Probe for ipaH gene:

CY5 5'-CACG GCCGAAGCTATGGTCAGAAGCCGTG -3'-DABCYL

A pair of primers for the rfbE gene:

rfbE-L: 5'-AGGTGAAGGTGGAATGGTTGTC-3'

rfbE-R: 5'-GCTTGTTCTAACTGGGCTAATC-3'

Probes for the rfbE gene:

5'-FAMC GGCCAAGGATTAGCTGTACATAGGC CG-DABCYL-3'

The underlined part is the target sequence of the improved molecular beacon probe.

Multiplex Fluorescent PCR-Modified Molecular Beacons for Detection of Foodborne Pathogens is a dual, triple, quadruple or multiplex fluorescent PCR-Modified Molecular Beacons for detection of foodborne pathogens.

The method for detecting food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons further includes the steps of sample processing and template extraction.

The samples include food samples, feces, and vomit specimens, wherein the processing of feces, vomit specimens and extraction templates is based on the amount of feces and vomit, suspended with 100-200 μL of normal saline, boiled, centrifuged at 10,000 rpm for 2 minutes, and 5 μL of The clear liquid can be used for real-time PCR reaction; the processing of food samples and the extraction of templates are to enrich the food samples in the enrichment solution for the bacteria to be tested, then take a total of 1.2mL of the enrichment solution and centrifuge at 10000rpm for 5 minutes, then discard it. After clear solution, add 30uL three-distilled water to boil, then take 5μL supernatant for real-time PCR reaction; or add lysate to lyse, extract with phenol-chloroform, and after ethanol precipitation, add 20μL water to dissolve, take 5μL solution For real-time PCR reactions.

The beneficial effects of the present invention are: on the basis of the improved molecular beacon technology, the present invention establishes a method for multiple real-time PCR to simultaneously detect multiple bacteria, the method:

(1) Random double, triple, quadruple or multiple combinations can be used to test multiple pathogenic bacteria at the same time; (2) High sensitivity, 32-100cfu/mL bacteria can be detected; (3) Strong specificity, There is no cross-reaction with more than ten kinds of bacteria in the control group; (4) The operation is simple, and the result observation is intuitive and clear, and 96 samples (including negative and positive controls) can be tested at one time; (5) The detection speed is fast, and the detection of stool and vomit samples only requires 2 hours testing time, it only takes 1-2 days to test food samples (including pre-treatment of samples).

Description of drawings

Figures 1 to 3 are the detection curves of fluorescent PCR-improved molecular beacons of the present invention.

Detailed ways

Taking the detection of food-borne pathogenic bacteria as an example, the present invention can be randomly combined to analyze the detection method of multiple fluorescent PCR-improved molecular beacons, so as to realize rapid and accurate detection of multiple pathogenic bacteria at the same time.

The improved molecular beacon probe is proposed on the basis of the original molecular beacon principle in order to improve the hybridization efficiency. The outstanding feature of the modified molecular beacon is that the arm part of the molecular beacon is also used as the target recognition sequence, not just an irrelevant added sequence for forming the hairpin structure. For the detection target sequence of the same length, the improved molecular beacon is shorter than the molecular beacon, and because the arm sequence is no longer suspended, it binds to the target sequence more tightly. Compared with molecular beacons, improved molecular beacons are easier to design and have a higher success rate. At the same time, the requirements for amplification conditions are not strict. Therefore, for the simultaneous detection of multiple target sequences, the advantages of improved molecular beacons are more obvious.

According to the conserved sequences of toxin genes or invasion genes of food-borne pathogens published by GenBank, namely, ctxA of Vibrio cholerae, TDH of Vibrio parahaemolyticus, invA of Salmonella, ipaH of Shigella, and ipaH of Staphylococcus aureus The hly gene of NUC and Listeria monocytogenes, the hemolysin gene rfbE of O157:H7 Escherichia coli, respectively designed primers and improved molecular beacon probes, and labeled the probes with different fluorescent agents. As a control, an improved molecular beacon-fluorescence PCR reaction system for detecting food-borne pathogens was established. And this detection system was applied to the rapid diagnosis of common bacterial food poisoning. The method for detecting food-borne pathogenic bacteria by multiplex fluorescent PCR mainly comprises the following steps:

(1) Design primers and probes respectively according to the conserved sequences of toxin genes or invasion genes of food-borne pathogens published by GenBank;

(2) Sample processing and template extraction to be tested;

(3) fluorescent PCR amplification;

(4) Detect the fluorescent signal, and use the Ct value of the number of cycles required when reaching the set threshold as the criterion for judging the result.

In step (1), according to the Vibrio cholerae enterotoxin gene ctxA sequence published by GenBank, the thermostable direct hemolysin gene TDH sequence of Vibrio parahaemolyticus, the thermostable nuclease gene NUC of Staphylococcus aureus, and the sequence of Listeria monocytogenes The sequence of the hemolysin gene hly, the sequence of the invasion gene ivnA of Salmonella, the sequence of the coding invasion plasmid antigen H gene ipaH of Shigella, and the sequence of the hemolytic gene rfbE of O157:H7 Escherichia coli, the primers of the improved molecular beacon of the present invention and the probes are:

A pair of primers for ctxA gene:

ctxA-F: 5′-TCCGGAGCATAGAGCTTGGA-3′,

ctxA-R: 5′-TCGATGATCTTGGAGCATTCC-3′

Probes for the ctxA gene:

HEX-5'- CCGTGGATTCATCATGCACCGCC ACGG-3'Dabcyl,

A pair of primers for TDH gene:

TDH-F: 5'-AAACATCTGCTTTTGAGCTTCCA-3',

TDH-R: 5′-CTCGAACAACAAACAATATCTCATCATCAG-3′,

Probes for the TDH gene:

FAM5′-CCGGGG TGTCCCTTTTTCCTGCCCCCGG -Dabcyl,

A pair of primers for NUC gene:

NUC-F: 5'-GGCAATACGCAAAGAGGTT-3'

NUC-L: 5'-CTTCTTCTATTTACGCCGTTATC-3'

Probes for NUC genes:

ROX-5'-CGATGCA GTCTAAGTAGCTCAGCAAATGCATC G-3'-DABCYL

A pair of primers for the hly gene:

hly-F: 5'-TGCAAGTCCTAAGACGCCA-3'

hly-L: 5'-CACTGCATCTCCGTGGTATACTAA-3'

Probes for the hly gene:

FAM-5'-CGCG CTTGTATATACTTATCGATTTCATCCGCGCG -3'-DABCYL

A pair of primers for the invA gene:

invA-F: 5'-GCGGAATATC(I)ATGACGCAGCT-3'

invA-L: 5'-CGCTACGTTTTGCTTCACGG-3'

Probes for the invA gene:

5'-FAM-CCG CCATTTGTATTGGTTGTTACGGC GG-DABCYL-3'

A pair of primers for ipaH gene:

ipaH-F: 5'-TGAAGGAAATGCGTTTCTATG-3'

ipaH-L: 5'-AGGGAGAACCAGTCCGTAAA-3'

Probe for ipaH gene:

CY5 5'-CACG GCCGAAGCTATGGTCAGAAGCCGTG -3'-DABCYL

A pair of primers for the rfbE gene:

rfbE-L: 5'-AGGTGAAGGTGGAATGGTTGTC-3'

rfbE-R: 5'-GCTTGTTCTAACTGGGCTAATC-3'

Probes for the rfbE gene:

5'-FAMC GGCCAAGGATTAGCTGTACATAGGC CG-DABCYL-3'.

The underlined part is the target sequence of the improved molecular beacon probe.

Step (2) sample processing and template extraction, the scope of application of the sample includes food samples, feces, vomit and other specimens. For feces and vomit samples, according to the amount of feces and vomit, suspend with 100-200 μL of normal saline, boil, centrifuge at 10,000 rpm for 2 minutes, and take 5 μL of supernatant for real-time PCR reaction. The processing of food samples and the extraction of templates are to enrich the food samples in the enrichment solution for the bacteria to be tested, take a total of 1.2mL of the enrichment solution and centrifuge at 10000rpm for 5 minutes, discard the supernatant, and add 30uL triple steamed Boil water, then take 5μL supernatant for real-time PCR reaction, or discard the supernatant and add lysate to lyse, extract with phenol-chloroform, and after ethanol precipitation, add 20μL water to dissolve, take 5μL solution for Real-time PCR reaction. Therefore, multiple templates will be formed when multiplex fluorescent PCR-modified molecular beacons are used to detect food samples.

The fluorescent PCR amplification reaction system of step (3) is:

1×PCR buffer

MgCl ₂ 0.5～5mmol

Each dNTP 0.05～1.5mmol

Each pair of primers 0.1～1.0μmol+0.1～1.0μmol

Each probe 0.1～1.0μmol

Taq enzyme 0.2～10U

Template 1～20μL

Total volume 20～100μL

The above reagent components: 1×PCR buffer, MgCl ₂ , Taq enzyme, and dNTP were all purchased from China Dalian Biotech Company.

The reaction conditions are: pre-denaturation at 90-100°C for 3-10 minutes, denaturation at 92-95°C for 15-60 seconds in 40-50 cycles, annealing at 50-60°C for 15-60 seconds, and extension at 72°C for 15-120 seconds.

Step (4) detects the Ct value of the fluorescent signal by using iCycler real-time PCR amplification instrument (Bio-Rad company) or MX4000 fluorescent PCR amplification instrument or Rotor fluorescent PCR amplification instrument to detect fluorescence during the annealing stage, and 96 samples can be detected at one time (including yin and yang controls). Judgment results based on the Ct value displayed by the fluorescent PCR amplification instrument: a Ct value of 0 or 40 means negative; a Ct value less than 38 means positive; when the Ct is between 38-40, the sample needs to be retested. It only takes 2 hours to detect stool and vomit samples, and only 1-2 days to detect food samples (including the pre-treatment of samples).

Example 1

In this example, the detection of Salmonella and Shigella by dual fluorescent PCR-improved molecular beacons is taken as an example to analyze the method for the detection of foodborne pathogens by multiplex fluorescent PCR-improved molecular beacons. According to the sequences of Salmonella invasion gene (ivnA) and Shigella invasion plasmid antigen H gene (ipaH) published by GenBank and comparative analysis, a pair of primers and probes were designed respectively as described above.

Test strains: 132 strains of 14 kinds of bacteria. Including 1 strain of enterotoxigenic E. coli (ETEC), 1 strain of invasive E. coli (EIEC), 1 strain of O157: H7 E. coli (O157: H7), 1 strain of pathogenic E. coli Enteropathogenic E.coli (EPEC), 1 strain of Citrobacter, 1 strain of Escherichia coli, 1 strain of Vibrio parahaemolyticus (V.parahaemolyticus), 1 strain of cholera (V.cholera ) Vibrio, 1 strain of Staphylococcus aureus (S.aureus), 1 strain of Listeria monocytogenes (Listeria monocytogenes), 1 strain of Bacillus cereus (B.cereus), 1 strain of Proteus, 70 strains of Salmonella (various serotypes), 50 strains of Shigella (various serotypes). All strains were isolated and identified in accordance with the National Inspection Standard of "Food Microbiology" promulgated by the People's Republic of China.

The method for simultaneously detecting Salmonella and Shigella using the above-mentioned fluorescent PCR method also includes the following steps:

(1) Sample processing and template extraction: the scope of application of samples includes food samples, feces, vomit and other specimens; the processing and extraction of feces and vomit specimens are the same as the above method; for food samples, when detecting Salmonella, 25g of food Place in 225mLSC to enrich bacteria for 10 hours; for Shigella, put 25g food in 225mLGN to enrich bacteria for 10 hours. , add 30uL triple-distilled water to boil, and then take 5μL supernatant to be used for real-time PCR reaction.

(2) Fluorescent PCR amplification, preparation of multiple fluorescent PCR reaction systems:

1×PCR buffer

_MgCl2 1.5mmol

dNTP each 0.05mmol

Primer for invA gene 0.2μmol+0.2μmol

Primer for ipaH gene 0.2μmol+0.2μmol

Probe for invA gene 0.2μmol

Probe for ipaH gene 0.2μmol

Taq enzyme 1U

Template 10 μL

Total volume 25μL

Real-time PCR reaction: pre-denaturation at 95°C for 5 minutes, 45 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds.

(3) Detection: iCycler real-time PCR amplification instrument (Bio-Rad Company) was used to detect fluorescence during the annealing stage, and 96 samples (including negative and positive controls) could be detected at one time. Judgment of results: Judging the results according to the Ct value displayed by the fluorescent PCR amplification instrument: Ct value 0 or 40: negative; Ct less than 38: positive; Ct between 38-40: the sample needs to be re-tested.

A total of 132 bacterial strains of the aforementioned 14 kinds of bacteria are detected by the improved molecular beacon system of the present invention, only Salmonella containing the ivnA gene and Shigella containing the ipaH gene have fluorescent signals, and other bacteria have no fluorescent signals, which further proves the effectiveness of fluorescent PCR. See Table 1 for specificity.

Table 1 Specificity analysis of dual real-time PCR for Salmonella and Shigella bacteria Detection quantity Number of positive invA real-time PCR tests Number of ipaH real-time PCR tests positive salmonella 70 70 0 Shigella 50 0 50 Toxigenic Escherichia coli 1 0 0 Pathogenic Escherichia coli 1 0 0 Invasive Escherichia coli 1 0 0 Escherichia coli 1 0 0 Citrobacter 1 0 0 cholera 1 0 0 Vibrio parahaemolyticus 1 0 0 O157: H7 Escherichia coli 1 0 0 Bacillus cereus 1 0 0 Proteus 1 0 0 Listeria monocytogenes 1 0 0 Staphylococcus aureus 1 0 0

It can be seen from Table 1 that the fluorescent PCR-improved molecular beacon of the present invention has strong specificity and has no cross-reaction with more than ten kinds of bacteria in the control group.

Carry out experiment with total 132 bacterial strains of aforementioned 14 kinds of bacteria, detect respectively the blank system that does not contain Salmonella or Shigella, the system that contains Salmonella and Shigella, A system containing Salmonella, and a system containing Shigella. The iCycler real-time PCR amplification instrument was used to detect the fluorescence in the annealing stage, and the fluorescence signal curve is shown in Figure 1. Among them, Fig. 1 (a) is the fluorescence signal curve measured by the fluorescent PCR amplification instrument of the blank system, Fig. 1 (b) is the fluorescence signal curve detected by multiplex fluorescence PCR containing Salmonella and Shigella, Fig. 1 (c) is the fluorescence signal curve The fluorescent signal curve of PCR detection containing Salmonella, Fig. 1(d) is the fluorescent signal curve of fluorescent PCR detection containing Shigella. From the comparative analysis in Figure 1, the multiplex fluorescent PCR detects multiple bacteria without cross-reaction, mutual interference and inhibition, no weakening of the fluorescent signal, and strong specificity.

Sensitivity analysis: 1 strain of Salmonella and 1 strain of Shigella were selected as representative strains for sensitivity analysis. Sensitivity analysis method of Salmonella bacteria liquid: After the Salmonella strains were cultured with SC enrichment liquid overnight, they were diluted 10 times, and a total of 10 dilutions were made, and then 1 mL of the bacteria liquid of 10 dilutions were sequentially taken for real-time PCR reaction. At the same time, the total number of bacteria is counted correspondingly according to the "Food Microbiological Inspection Standard" promulgated by the People's Republic of China. Sensitivity analysis of Shigella bacterial fluid: After Shigella strains were cultured with broth enrichment fluid overnight, the rest of the steps were operated according to the method of "Sensitivity Analysis of Salmonella bacterial fluid". The result shows that the fluorescence PCR method of the present invention has high sensitivity, and bacteria of 32-100 cfu/mL can be detected.

Repeatability test: Repeat 10 times of fluorescent PCR amplification for 1 strain of Salmonella and 1 strain of Shigella respectively, and the difference in Ct value for 10 times is less than 0.1 cycle.

A total of 350 samples were collected, including food samples and food poisoning samples. 350 samples were detected by fluorescent PCR and verified by traditional bacterial culture method. According to the above method, the sample was processed and the template was extracted for detection. The comparison between the traditional detection method and the fluorescent PCR detection method is shown in Table 2.

Table 2 Comparison of traditional detection methods and fluorescent PCR detection methods Test items Sample category Number of samples fluorescent PCR traditional method positive number detection time positive number detection time salmonella poop 50 30 2 hours 30 4 days food 200 65 1 day 32 4 days Shigella poop 50 1 2 hours 1 4 days food 100 0 1 day 0 4 days

The multiplex fluorescent PCR method only takes 2 hours to detect stool and vomit samples, and only 1 day to detect food samples (including the pre-treatment of samples), and can detect multiple bacteria at the same time. Therefore, the multiple fluorescent PCR method saves time and labor, has high accuracy, and can meet the rapid diagnosis of diseases. Moreover, the coincidence rate of the results of the fluorescent PCR method and the traditional detection method is 100%, and the sensitivity of the fluorescent PCR method is higher than that of the traditional detection method, and the bacteria can be detected if the sample contains 100cfu/mL of bacteria.

Example 2

In this example, the detection of Shigella, O157:H7 Escherichia coli and Listeria monocytogenes by triple fluorescent PCR-improved molecular beacon is taken as an example. According to GenBank, the sequence of the coding invasion plasmid antigen H gene (ipaH) of Shigella, the sequence of the hemolysin gene rfbE of Escherichia coli O157:H7, and the sequence of the hemolysin gene (hly) of Listeria monocytogenes were published, respectively The designed pair of primers and probes and their amplified fragments are the same as those described above.

Test strains: O157: H7 Escherichia coli (O157: H7), Listeria monocytogenes, Shigella (various serotypes). All strains were isolated and identified in accordance with the National Inspection Standard of "Food Microbiology" promulgated by the People's Republic of China.

The method for simultaneously detecting Shigella, O157:H7 Escherichia coli, and Listeria monocytogenes by utilizing the above-mentioned fluorescent PCR method also includes the following steps:

(1) Sample processing and template extraction: the scope of application of samples includes food samples, feces, vomit and other specimens; the processing and extraction methods of feces and vomit specimens are the same as Example 1; food samples: for Shigella, 25g of food Put it in 225mLGN for 10 hours, for O157:H7 Escherichia coli, put 25g of food in 225mL intestinal enrichment solution for 10 hours, for Listeria monocytogenes: put 25g of food in 225mLLB1 Bacteria for 24 hours, after enrichment, take 0.4mL of enrichment solution, a total of 1.2mL, centrifuge at 10000rpm for 5 minutes, discard the supernatant, add lysate to lyse, extract with phenol-chloroform, and after ethanol precipitation, add 20μL Dissolve in water, and take 5 μL of the solution for real-time PCR reaction.

(2) Reaction system for triple fluorescent PCR amplification:

1×PCR buffer

_MgCl2 1.5mmol

dNTP each 0.05mmol

Primer for ipaH gene 0.2μmol+0.2μmol

Primer for rfbE gene 0.2μmol+0.2μmol

Primer for hly gene 0.2μmol+0.2μmol

Probe for ipaH gene 0.2μmol

rfbE gene probe 0.2μmol

Probe for hly gene 0.2μmol

Taq enzyme 1U

Template 10 μL

Total volume 25μL

Real-time PCR reaction: pre-denaturation at 95°C for 5 minutes, 45 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds.

(3) Detection: Rotor fluorescent PCR amplification instrument (Roche Company) was used to detect fluorescence during the annealing stage, and 96 samples (including negative and positive controls) could be detected at one time. Judgment of results: Judging the results according to the Ct value displayed by the fluorescent PCR amplification instrument: Ct value 0 or 40: negative; Ct less than 38: positive; Ct between 38-40: the sample needs to be re-tested.

The system containing Shigella, O157:H7 Escherichia coli, and Listeria monocytogenes is detected by the triple fluorescent PCR method of the present invention, and the fluorescence in the annealing stage is detected by the iCycler real-time PCR amplification instrument, and three fluorescence signal curves are simultaneously measured as follows: figure 2. Wherein, Fig. 2(a) is the fluorescent signal curve measured by the fluorescent PCR amplification instrument of the Shigella system, and the number of curves indicates the number of samples. Figure 2(b) is the fluorescence signal curve of O157:H7 Escherichia coli, and Figure 2(c) is the fluorescence signal curve of the detection of Listeria monocytogenes by fluorescent PCR. From the comparative analysis in Figure 2, the triple fluorescent PCR detects a variety of bacteria without cross-reaction and strong specificity.

The multiplex fluorescent PCR method only needs 2 hours to detect stool and vomit samples, and only 1-2 days to detect food samples (including the pre-treatment of samples), and can detect multiple bacteria at the same time. Therefore, the multiple fluorescent PCR method saves time and labor, has high accuracy, and can meet the rapid diagnosis of diseases. Moreover, the coincidence rate of the results of the fluorescent PCR method and the traditional detection method is 100%, and the sensitivity of the fluorescent PCR method is higher than that of the traditional detection method, and the bacteria can be detected if the sample contains 100cfu/mL of bacteria.

Example 3

This example illustrates a method for the simultaneous detection of Shigella, Vibrio cholerae, Staphylococcus aureus, and Escherichia coli O157:H7 by quadruple fluorescent PCR-modified molecular beacons. According to GenBank, the sequence of the coding invasion plasmid antigen H gene (ipaH) of Shigella, the sequence of the enterotoxin gene ctxA of Vibrio cholerae, the sequence of the thermostable nuclease gene NUC of Staphylococcus aureus, and the sequence of Escherichia coli O157:H7 were released according to GenBank. The rfbE sequence of the hemolysin gene, a pair of primers and probes and amplified fragments designed respectively are the same as those described above.

Test strains: Shigella, Vibrio cholerae, Staphylococcus aureus, Escherichia coli O157:H7. All strains were isolated and identified in accordance with the National Inspection Standard of "Food Microbiology" promulgated by the People's Republic of China.

The method for simultaneously detecting Shigella, Vibrio cholerae, Staphylococcus aureus, and O157:H7 Escherichia coli using the above-mentioned fluorescent PCR method also includes the following steps:

(1) Sample processing and template extraction: the scope of application of samples includes food samples, feces, vomit and other specimens; the processing and extraction methods of feces and vomit specimens are the same as Example 1 and Example 2; for food samples: for Shigella , put 25g food in 225mL GN for 10 hours, for O157:H7 Escherichia coli, put 25g food in 225mL intestinal enrichment solution for 10 hours, for Staphylococcus aureus: put 25g food in 225mL Enrichment in 7.5% NaCl for 10 hours. For Vibrio cholerae, put 25g of food in 225mL of alkaline peptone water for enrichment, then take 0.3mL of each enrichment solution, a total of 1.2mL, centrifuge at 10000rpm for 5 minutes, discard the supernatant, Add lysate to lyse, extract with phenol-chloroform, and precipitate with ethanol, add 20 μL of water to dissolve, and take 5 μL of the solution for real-time PCR reaction.

(2) Quadruple fluorescent PCR amplification, preparation of quadruple fluorescent PCR reaction system:

1×PCR buffer

MgCl2 1.5mmol

dNTP each 0.05mmol

Primer for ipaH gene 0.2μmol+0.2μmol

Primer for ctxA gene 0.2μmol+0.2μmol

Primer for NUC gene 0.2μmol+0.2μmol

Primer for rfbE gene 0.2 μmol

Probe for ipaH gene 0.2μmol

ctxA gene probe 0.2μmol

Probe for NUC gene 0.2μmol

Probe for rfbE gene 0.2μmol

Taq Enzyme 1U

Template 10 μL

Total volume 25μL

The conditions of the real-time PCR reaction were the same as those in Example 1 and Example 2.

(3) Detection: Rotor fluorescent PCR amplification instrument (Roche Company) was used to detect fluorescence during the annealing stage, and 96 samples (including negative and positive controls) could be detected at one time. Judgment of results: Judging the results according to the Ct value displayed by the fluorescent PCR amplification instrument: Ct value 0 or 40: negative; Ct less than 38: positive; Ct between 38-40: the sample needs to be re-tested.

The system containing Shigella, Vibrio cholerae, Staphylococcus aureus and O157:H7 Escherichia coli is detected by the quadruple fluorescent PCR method of the present invention. The iCycler real-time PCR amplification instrument was used to detect the fluorescence in the annealing stage, and the fluorescence signal curve is shown in Figure 3. Wherein, Fig. 3(a) is the fluorescent signal curve measured by the fluorescent PCR amplification instrument of the Shigella system, and the number of curves indicates the number of samples. Figure 3(b) is the fluorescence signal curve of Vibrio cholerae, Figure 3(c) is the fluorescence signal curve of Staphylococcus aureus, and Figure 3(d) is the fluorescence signal curve of O157:H7 Escherichia coli. From the comparative analysis in Figure 3, the quadruple fluorescent PCR detects multiple bacteria without cross-reaction and strong specificity.

Various other foodborne pathogens such as Salmonella, Shigella, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Proteus, Hemolytic Streptococcus, Vibrio parahaemolyticus, and Vibrio cholerae Bacteria, etc., were randomly combined by double, triple, quadruple, etc., and detected by the above-mentioned multiple fluorescent PCR-improved molecular beacon detection method, using iCycler real-time PCR amplification instrument (Bio-Rad company) to detect fluorescence during the annealing stage, and various food The detection of the derived pathogenic bacteria all showed highly specific curves similar to the aforementioned fluorescence signal curves, and the detection sensitivity was high.

The detection system of multiple fluorescent PCR-improved molecular beacons for the detection of foodborne pathogens can be developed into a sequence of rapid detection kits to meet the daily inspection requirements of disease prevention and control agencies, inspection and quarantine departments, and technical supervision departments and public health emergencies. Rapid response has significant social and economic benefits.

Claims (9)

1. A method for multiple fluorescent PCR-improved molecular beacons to detect food-borne pathogenic bacteria, comprising the following steps: (1) Design multiple pairs of primers and multiple improved molecular beacon probes respectively according to the specific gene sequences of various pathogenic bacteria to be tested; (2) Prepare a reaction system for fluorescent PCR-improved molecular beacon amplification gene sequence; (3) using the various fluorescent PCR-improved molecular beacons of step (1) to simultaneously amplify the sequence of multiple pathogenic bacteria specific genes to be tested; (4) Using the hybridization between the molecular beacon and the amplification product to detect the fluorescence intensity of the reaction system, the Ct value of the number of cycles required to reach the set threshold is used as the result judgment standard, and the Ct value is 0 or 40: negative; Ct less than 38: Positive. 2. The method for detecting food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons as claimed in claim 1, characterized in that: the reaction system of the fluorescent PCR-improved molecular beacons to amplify gene sequences is: 1×PCR buffer MgCl ₂ 0.5～5mmol Each dNTP 0.05～1.5mmol Each pair of primers 0.1～1.0μmol+0.1～1.0μmol Each probe 0.1～1.0μmol Taq enzyme 0.2～10U Template 1～20μL Total volume 20～100μL 3. The method for detecting food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons as claimed in claim 1, characterized in that: the reaction condition for the amplification of specific gene sequences by said fluorescent PCR-improved molecular beacons is 90 Pre-denaturation at ~100°C for 3-10 minutes, denaturation at 92-95°C for 15-60 seconds in 40-50 cycles, annealing at 50-60°C for 15-60 seconds, extension at 70-72°C for 15-120 seconds. 4. The method for detecting foodborne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons according to any one of claims 1 to 3, characterized in that: the pathogenic bacteria are selected from Vibrio cholerae, para A combination of one or more of Vibrio hemolyticus, Staphylococcus aureus, Listeria monocytogenes, Salmonella, Shigella, and Escherichia coli O157:H7. 5. The method for detecting foodborne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons as claimed in claim 4, characterized in that: the specific gene sequences of the pathogenic bacteria are respectively the ctxA sequence of the Vibrio cholerae enterotoxin gene , heat-resistant direct hemolysin gene TDH sequence of Vibrio parahaemolyticus, thermostable nuclease gene NUC of Staphylococcus aureus, sequence of hemolysin gene hly of Listeria monocytogenes, invasion gene ivnA sequence of Salmonella, Shigella Bacteria coding invasion plasmid antigen H gene ipaH sequence, and O157: H7 Escherichia coli hemolysin gene rfbE sequence. 6. The multiple fluorescent PCR-improved molecular beacon method for detecting food-borne pathogenic bacteria as claimed in claim 5, characterized in that: the primers and probes of the improved molecular beacon are respectively: A pair of primers for ctxA gene: ctxA-F: 5′-TCCGGAGCATAGAGCTTGGA-3′, ctxA-R: 5′-TCGATGATCTTGGAGCATTCC-3′ Probes for the ctxA gene: HEX-5'- CCGTGGATTCATCATGCACCGCC ACGG-3'Dabcyl, A pair of primers for TDH gene: TDH-F: 5'-AAACATCTGCTTTTGAGCTTCCA-3', TDH-R: 5′-CTCGAACAACAAACAATATCTCATCATCAG-3′, Probes for the TDH gene: FAM5′-CCGGGG TGTCCCTTTTTCCTGCCCCCGG -Dabcyl, A pair of primers for NUC gene: NUC-F: 5'-GGCAATACGCAAAGAGGTT-3' NUC-L: 5'-CTTCTTCTATTTACGCCGTTATC-3' Probes for NUC genes: ROX-5'-CGATGCA GTCTAAGTAGCTCAGCAAATGCATC G-3'-DABCYL A pair of primers for the hly gene: hly-F: 5'-TGCAAGTCCTAAGACGCCA-3' hly-L: 5'-CACTGCATCTCCGTGGTATACTAA-3' Probes for the hly gene: FAM-5'-CGCG CTTGTATATACTTATCGATTTCATCCGCGCG -3'-DABCYL A pair of primers for the invA gene: invA-F: 5'-GCGGAATATC(I)ATGACGCAGCT-3' invA-L: 5'-CGCTACGTTTTGCTTCACGG-3' Probes for the invA gene: 5'-FAM-CCG CCATTTGTATTGGTTGTTACGGC GG-DABCYL-3' A pair of primers for ipaH gene: ipaH-F: 5'-TGAAGGAAATGCGTTTCTATG-3' ipaH-L: 5'-AGGGAGAACCAGTCCGTAAA-3' Probe for ipaH gene: CY5 5'-CACG GCCGAAGCTATGGTCAGAAGCCGTG -3'-DABCYL A pair of primers for the rfbE gene: rfbE-L: 5'-AGGTGAAGGTGGAATGGTTGTC-3' rfbE-R: 5'-GCTTGTTCTAACTGGGCTAATC-3' Probes for the rfbE gene: 5'-FAMC GGCCAAGGATTAGCTGTACATAGGC CG-DABCYL-3', where the underlined part is the target sequence of the improved molecular beacon probe. 7. The method for detecting food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons as claimed in claim 1, characterized in that: the detection of food-borne pathogenic bacteria by multiple fluorescent PCR-improved molecular beacons is double , Triple or Quadruple Fluorescent PCR-Modified Molecular Beacons for Detection of Foodborne Pathogens. 8. The method for detecting food-borne pathogenic bacteria by multiplex fluorescent PCR-improved molecular beacons as claimed in claim 1, further comprising the steps of sample processing and template extraction. 9. The method for detecting food-borne pathogenic bacteria by multiplex fluorescent PCR-improved molecular beacons as claimed in claim 8, characterized in that: said samples include food samples, feces, or vomit specimens, wherein feces, vomit According to the amount of feces and vomitus, suspend with 100-200 μL of normal saline, boil, centrifuge at 10,000 rpm for 2 minutes, and take 5 μL of supernatant for real-time PCR reaction; the processing of food samples and the extraction of templates are After enriching the food samples in the enrichment solution for the bacteria to be tested, take a total of 1.2mL of the enrichment solution and centrifuge at 10,000rpm for 5 minutes, discard the supernatant, add 30uL of triple-distilled water to boil, and then take 5μL of the supernatant. It can be used for real-time PCR reaction, or discard the supernatant and add lysate to lyse, extract with phenol-chloroform, and after ethanol precipitation, add 20 μL of water to dissolve, and take 5 μL of the solution for real-time PCR reaction.