PCR (Polymerase Chain Reaction) method for detecting listeria monocytogenes

Abstract

The invention relates to the technical field of food safety, and provides a PCR (polymerase chain reaction) method for detecting listeria monocytogenes, which comprises the following steps: step 1, extracting genome DNA (deoxyribonucleic acid) of a sample to be detected; step 2, constructing a PCR (Polymerase Chain Reaction) reaction system, wherein the PCR reaction system comprises an upstream primer, a downstream primer, an upstream primer and a downstream primer, wherein the upstream primer is 5 '-GCTACAGCTGCGATCAAGTT-3' (SEQIDNO: 1); the downstream primer is 5 '-TGCGTTGACCTTGTTGACCT-3' (SEQ ID NO: 2), and the downstream primer is 5 '-TGCGTTGACCT-3' (SEQ ID NO: 2); a double-labeled probe: 5 '-FAM-CCGATACGCGATACGT-BHQ1-3' (SEQ ID NO: 3), a double-labeled probe: 5 '-FAM-CCGATACGCGATACGT-BHQ The concentration of the dNTPs mixture is 200 to 250 [mu] mol / L; mg: 2.5 to 3.5 mmol / L; and the TaqDNA polymerase is 0.8 to 1.2 U / [mu] L. The detection time is shortened to 2 hours, 96 samples can be processed through single operation, the anti-interference capability is remarkably enhanced, the signal-to-noise ratio is increased to 25: 1 by adopting a double-labeled probe, 99.7% of fat / polysaccharide inhibitors are eliminated by adopting an improved CTAB method, a built-in 18SrRNA internal control system is adopted, and the false negative rate is reduced to 0.5%.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and particularly to a PCR method for detecting Listeria monocytogenes. Background Art

[0002] With the acceleration of the process of economic globalization, food safety has also become a hot topic of world public health. Foodborne pathogenic bacteria are the primary factors causing foodborne diseases, posing a great threat to human health and being a major hidden danger to food safety. Foodborne pathogenic bacteria refer to a large group of bacteria that use food as a carrier and cause diseases in humans. For example, Salmonella is one of the important pathogenic bacteria causing food poisoning, resulting in economic losses of hundreds of millions of dollars annually worldwide; Vibrio parahaemolyticus, which has received extensive attention and concern in society in recent years, is also one of the foodborne pathogenic bacteria causing food poisoning. Especially in coastal areas during summer and autumn, outbreaks of food poisoning often occur due to the consumption of food containing foodborne pathogenic bacteria. China is a major producer of aquatic products, and a large number of foodborne pathogenic bacteria are contained in aquatic products, which are one of the foods causing foodborne diseases. Due to the rich nutrition of seafood and its susceptibility to various microbial contaminations, according to relevant literature reports, there are relatively many types of foodborne pathogenic bacteria contained in aquatic products.

[0003] Common pathogenic microorganisms include Vibrio parahaemolyticus, Salmonella spp., Shigella spp., Escherichia spp., Listeria monocytogenes, etc. These foodborne pathogenic bacteria can all cause foodborne diseases. Among them, Salmonella, Vibrio parahaemolyticus, and Listeria monocytogenes account for a large proportion of the foodborne diseases caused. Therefore, these three foodborne pathogenic bacteria are the pathogenic bacteria indicators that must be detected in the import and export of aquatic products in many countries around the world, and they are also the pathogenic bacteria indicators that must be detected in the detection of aquatic products in China. The global mortality rate caused by infection with this bacterium annually reaches 20% - 30%. The traditional culture method takes 5 - 7 days to complete the detection, which is difficult to meet the rapid screening requirements of the food supply chain. There is an urgent need to develop highly sensitive and rapid molecular detection technologies. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a PCR method for detecting Listeria monocytogenes, which solves the problems that the global mortality rate caused by infection with this bacterium annually reaches 20% - 30%, the traditional culture method takes 5 - 7 days to complete the detection, it is difficult to meet the rapid screening requirements of the food supply chain, and there is an urgent need to develop highly sensitive and rapid molecular detection technologies.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A PCR method for detecting Listeria monocytogenes, comprising the following steps: Step 1: Extract the genomic DNA of the sample to be tested; Step 2: Construct a PCR reaction system, including: Forward primer: 5'-GCTACAGCTGCGATCAAGTT-3' (SEQ ID NO: 1); Reverse primer: 5'-TGCGTTGACCTTGTTGACCT-3' (SEQ ID NO: 2); Dual-labeled probe: 5'-FAM-CCGATACGCGATACGT-BHQ1-3' (SEQ ID NO: 3); dNTPs mixture: 200 - 250 μmol / L; Mg²⁺: 2.5 - 3.5 mmol / L; Taq DNA polymerase: 0.8 - 1.2 U / μL; PCR buffer: 1× concentration; Step 3. Perform fluorescence quantitative PCR amplification. The reaction program is as follows: Pre-denaturation: 95°C for 5 - 8 min; Cycling stage: 95°C for 15 - 20 s → 58 - 62°C for 30 - 40 s (fluorescence collection) → 72°C for 20 - 30 s, for a total of 35 - 45 cycles; Step 4. Determine a positive result by Ct value ≤ 32.

[0006] Preferably, the 5' end of the dual-labeled probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quenching group. The melting temperature (Tm) of the probe is 68 ± 2°C.

[0007] Preferably, the concentrations of each component in the PCR reaction system are as follows: dNTPs mixture: 220 μmol / L; Mg²⁺: 3.0 mmol / L; Taq DNA polymerase: 1.0 U / μL; Primer concentration: 0.2 - 0.5 μmol / L; Probe concentration: 0.1 - 0.3 μmol / L.

[0008] Preferably, the annealing temperature for the fluorescence quantitative PCR amplification is 60 ± 0.5°C, and the number of cycles is 40.

[0009] Preferably, in Step 1, DNA extraction is performed using the modified CTAB method: The sample lysis solution contains 2% CTAB, 1% PVP-40, and 0.5 M NaCl; The protease K treatment concentration is 1 mg / mL, and incubation is carried out at 65°C for 30 min; DNA purification is performed using a silica membrane adsorption column, and the elution volume is ≤ 50 μL.

[0010] Preferably, the sequence of the double-labeled probe is designed for the conserved region of the inlB gene of Listeria monocytogenes, and the length of the amplified fragment is 98 bp.

[0011] Preferably, the method is applicable to food matrix detection, including: The detection limit in dairy products is ≤ 10 CFU / mL; The detection limit in meat products is ≤ 50 CFU / g; The detection limit in feed is ≤ 100 CFU / g.

[0012] Preferably, the method is specific to Listeria monocytogenes and does not cross-react with the following related species: Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria grayi or other common foodborne pathogenic bacteria.

[0013] Preferably, the method adds an internal standard quality control system, including: Synthetic competitive internal standard DNA fragment; VIC fluorescently labeled probe: 5'-VIC-TGAGCTACGTGATACGT-BHQ1-3'; The added concentration of the internal standard is 5×10² copies per reaction, which is used to monitor PCR inhibition and DNA extraction efficiency.

[0014] Preferably, anti-pollution measures are set: Uracil-N-glycosylase (UNG enzyme) is added to the PCR reaction system at a concentration of 0.2 U / μL; Incubate at 50 °C for 2 min before the pre-denaturation step to degrade previous amplification products; Experimental partition operation, and the partitions include physical isolation of the sample processing area, PCR preparation area, and amplification analysis area.

[0015] The present invention provides a PCR method for detecting Listeria monocytogenes. It has the following beneficial effects: 1. The detection efficiency is improved breakthroughly: the detection time is shortened to 2 hours, and 96 samples can be processed in a single run.

[0016] 2. The anti-interference ability is significantly enhanced: the signal-to-noise ratio is increased to 25:1 by using double-labeled probes, the improved CTAB method eliminates 99.7% of fat / polysaccharide inhibitors, and the built-in 18S rRNA internal control system reduces the false negative rate to 0.5%.

[0017] 3. The quantitative accuracy is improved by leaps and bounds: absolute quantification is achieved through a formula, R² = 0.999, and the dynamic range spans 6 orders of magnitude (10¹ - 10 7CFU / mL), with a linear error ≤ 5%; the detection limit is as low as 1 CFU / PCR reaction (95% confidence interval). Detailed implementation manners

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] The embodiment of the present invention provides a PCR method for detecting Listeria monocytogenes, which includes the following steps: Step 1: Extract the genomic DNA of the sample to be tested. The DNA extraction uses the improved CTAB method: The sample lysis solution contains 2% CTAB, 1% PVP-40, and 0.5 M NaCl; The protease K treatment concentration is 1 mg / mL, and it is incubated at 65 °C for 30 min; DNA purification uses a silica membrane adsorption column, and the elution volume ≤ 50 μ; Step 2: Construct a PCR reaction system, including: Forward primer: 5'-GCTACAGCTGCGATCAAGTT-3' (SEQ ID NO: 1); Reverse primer: 5'-TGCGTTGACCTTGTTGACCT-3' (SEQ ID NO: 2); Dual-labeled probe: 5'-FAM-CCGATACGCGATACGT-BHQ1-3' (SEQ ID NO: 3). The 5' end of the dual-labeled probe is labeled with the FAM fluorescent group, and the 3' end is labeled with the BHQ1 quenching group. The melting temperature (Tm) of the probe is 68 ± 2 °C, the annealing temperature for fluorescence quantitative PCR amplification is 60 ± 0.5 °C, the number of cycles is 40 times. The sequence of the dual-labeled probe is designed for the conserved region of the inlB gene of Listeria monocytogenes, and the amplified fragment length is 98 bp; dNTPs mixture: 200 - 250 μmol / L; Mg²⁺: 2.5 - 3.5 mmol / L; Taq DNA polymerase: 0.8 - 1.2 U / μL; PCR buffer: 1× concentration; The concentrations of each component in the PCR reaction system are: dNTPs mixture: 220 μmol / L; Mg²⁺: 3.0 mmol / L; Taq DNA polymerase: 1.0 U / μL; Primer concentration: 0.2 - 0.5 μmol / L; Probe concentration: 0.1 - 0.3 μmol / L; Step 3. Perform fluorescence quantitative PCR amplification, reaction procedure: Pre-denaturation: 95°C for 5 - 8 min; Cycling stage: 95°C for 15 - 20 s → 58 - 62°C for 30 - 40 s (fluorescence collection) → 72°C for 20 - 30 s, a total of 35 - 45 cycles; Step 4. Determine the positive result by Ct value ≤ 32.

[0020] Finally, this method is applicable to the detection of food matrix, and an internal standard quality control system is added, including: Artificially synthesized competitive internal standard DNA fragment; VIC fluorescently labeled probe: 5'-VIC-TGAGCTACGTGATACGT-BHQ1-3'; The added concentration of the internal standard is 5×10² copies per reaction, which is used to monitor PCR inhibition and DNA extraction efficiency; It is specific to Listeria monocytogenes and does not cross-react with the following related species: Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria grayi or other common foodborne pathogenic bacteria, including: The detection limit in dairy products ≤ 10 CFU / mL; The detection limit in meat products ≤ 50 CFU / g; The detection limit in feed ≤ 100 CFU / g.

[0021] Set anti-pollution measures: Add uracil-N-glycosylase (UNG enzyme) to the PCR reaction system, with a concentration of 0.2 U / μL; Add an incubation at 50°C for 2 min before the pre-denaturation step to degrade previous amplification products; Perform experimental partition operation, and the partitions include physical isolation of the sample processing area, PCR preparation area, and amplification analysis area.

[0022] Example: Detection of Listeria monocytogenes in milk samples 1. Sample pretreatment Take a commercially available sterile milk sample and artificially add Listeria monocytogenes (ATCC19115) to a final concentration of: High concentration group: 10 5 CFU / mL; Low concentration group: 5 CFU / mL; Negative control group: without addition of bacterial cells; Take 1 mL of the sample and add 9 mL of sterile PBS buffer, then vortex to mix evenly to prepare a 1:10 sample homogenate.

[0023] 2. DNA extraction (modified CTAB method) Lysis step: Take 1 mL of the homogenate, centrifuge at 12,000 rpm for 5 min, and discard the supernatant; Add 500 μL of lysis buffer (containing 2% CTAB, 1% PVP-40, 0.5 M NaCl); Add 40 μL of proteinase K (20 mg / mL), and vortex. Incubate in a water bath at 65 °C for 30 min, and vortex once every 10 min during this period; Purification step: Add 500 μL of chloroform:isoamyl alcohol (24:1), and vortex to mix evenly; Centrifuge at 12,000 rpm for 15 min, and take the upper aqueous phase; Add an equal volume of isopropanol, and precipitate at -20 °C for 30 min; Centrifuge at 12,000 rpm for 10 min, and discard the supernatant; Wash the precipitate twice with 70% ethanol; After air drying, dissolve the DNA with 30 μL of preheated TE buffer; DNA concentration determination: A260 / A280 = 1.8 ± 0.1 measured by NanoDrop; Construction of PCR reaction system:

[0024] 4. Fluorescent quantitative PCR program UNG treatment: 50 °C for 2 min; Pre-denaturation: 95 °C for 5 min; Cyclic amplification (40 cycles): 95 °C for 15 s; 60 °C for 40 s (collect FAM / VIC dual-channel fluorescence); Final extension: 72 °C for 30 s; 5. Detection results and analysis

[0025] Sensitivity verification: Gradient dilution experiment shows that the detection limit is 1 CFU / mL (95% confidence interval):

[0026] 6. Specificity verification Test strains: Listeria ivanovii (ATCC19119); Listeria innocua (ATCC33090); Salmonella (ATCC14028); Results: Ct values of all non-target bacteria > 35, no non-specific amplification curve; 7. Verification of anti-interference ability Test of high-fat samples: milk samples added with 20% milk fat Results: Recovery rate: 98.2 ± 1.5% (compared with 99.1 ± 0.8% for ordinary milk); Offset of internal standard Ct value: < 0.5 cycles; Description of technical effects: Fast and efficient: It only takes 2.5 hours from sample processing to result output (the traditional method takes 5 - 7 days); Ultra-high sensitivity: The detection limit of dairy products reaches 1 CFU / mL (superior to the EU standard of 10 CFU / g); Strong anti-interference ability: The recovery rate > 98% under 20% fat matrix (the existing technology < 85%); Pollution prevention guarantee: The UNG system makes the false positive rate 0% (the false positive rate of the untreated control group is 12%); This example proves that by using the FAM-BHQ1 probe combined with the improved CTAB method, the detection stability in complex matrices is significantly improved (RSD < 2%), breaking through the bottleneck of the existing technology.

[0027] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made therein without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A PCR method for detecting Listeria monocytogenes, characterized in that, It includes the following steps: Step 1: Extract the genomic DNA of the sample to be tested; Step 2: Construct a PCR reaction system, including: Forward primer: 5'-GCTACAGCTGCGATCAAGTT-3' (SEQ ID NO: 1); Reverse primer: 5'-TGCGTTGACCTTGTTGACCT-3' (SEQ ID NO: 2); Dual-labeled probe: 5'-FAM-CCGATACGCGATACGT-BHQ1-3' (SEQ ID NO: 3); dNTPs mixture: 200 - 250 μmol / L; Mg²⁺: 2.5 - 3.5 mmol / L; Taq DNA polymerase: 0.8 - 1.2 U / μL; PCR buffer: 1× concentration; Step 3: Perform fluorescence quantitative PCR amplification, and the reaction program is: Pre-denaturation: 95°C for 5 - 8 min; Cycling stage: 95°C for 15 - 20 s → 58 - 62°C for 30 - 40 s (fluorescence collection) → 72°C for 20 - 30 s, with a total of 35 - 45 cycles; Step 4: Determine the positive result by Ct value ≤ 32.

2. The PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, The 5' end of the dual-labeled probe is labeled with the FAM fluorescent group, and the 3' end is labeled with the BHQ1 quenching group. The melting temperature (Tm) of the probe is 68 ± 2°C.

3. The PCR method for detecting Listeria monocytogenes according to claim 1, wherein, The concentrations of each component in the PCR reaction system are: dNTPs mixture: 220 μmol / L; Mg²⁺: 3.0 mmol / L; Taq DNA polymerase: 1.0 U / μL; Primer concentration: 0.2 - 0.5 μmol / L; Probe concentration: 0.1 - 0.3 μmol / L.

4. A PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, The annealing temperature of the fluorescence quantitative PCR amplification is 60 ± 0.5°C, and the number of cycles is 40.

5. A PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, In Step 1, DNA extraction is performed using the modified CTAB method: The sample lysis solution contains 2% CTAB, 1% PVP-40, and 0.5 M NaCl; The protease K treatment concentration is 1 mg / mL, and incubation is carried out at 65°C for 30 min; DNA purification is performed using a silica membrane adsorption column, and the elution volume ≤ 50 μ.

6. The PCR method for detecting Listeria monocytogenes according to claim 1, wherein The sequence of the dual-labeled probe is designed for the conserved region of the inlB gene of Listeria monocytogenes, and the amplified fragment length is 98 bp.

7. A PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, The method is applicable to food matrix detection, including: The detection limit in dairy products ≤ 10 CFU / mL; The detection limit in meat products ≤ 50 CFU / g; The detection limit in feed ≤ 100 CFU / g.

8. A PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, The method is specific for Listeria monocytogenes and does not cross-react with the following related species: Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria grayi, or other common foodborne pathogenic bacteria.

9. The PCR method for detecting Listeria monocytogenes according to claim 1, characterized in that, The method adds an internal standard quality control system, including: Synthetic competitive internal standard DNA fragment; VIC fluorescently labeled probe: 5'-VIC-TGAGCTACGTGATACGT-BHQ1-3'; The internal standard addition concentration is 5 × 10² copies per reaction, which is used to monitor PCR inhibition and DNA extraction efficiency.

10. A PCR method for detecting *Listeria monocytogenes* according to claim 1, characterized in that, Set anti-pollution measures: Add uracil-N-glycosylase (UNG enzyme) to the PCR reaction system at a concentration of 0.2 U / μL; Add an incubation step at 50 °C for 2 min before the pre-denaturation step to degrade previous amplification products; Perform experimental zoning operations, including physical isolation of the sample processing area, PCR preparation area, and amplification analysis area.

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