A method for detecting foodborne pathogenic bacteria based on PMA-double fluorescent quantitative PCR

Abstract

The application provides a method for detecting foodborne pathogenic bacteria based on PMA-double fluorescent quantitative PCR, and belongs to the technical field of food microbiological detection. The method comprises the following steps: homogenizing baked food, enriching bacteria bodies through differential centrifugation, continuously treating twice with PMA with a final concentration of 10 μmol / L, inhibiting DNA amplification of damaged dead bacteria through blue light irradiation, and extracting DNA through complex lysis of lysozyme, lysostaphin and protease K; taking nuc gene and gyrB gene as targets, synchronously detecting Staphylococcus aureus and Bacillus cereus through HEX and FAM channels, and carrying out semi-quantification according to a food matrix standard curve. The detection range of the method is 10 2 ~ 10 7 CFU / g, the detection limit is 10 2 CFU / g, and the method is suitable for rapid detection of the two target bacteria in baked food.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology detection technology, and particularly relates to a method for detecting foodborne pathogens based on PMA-dual fluorescence quantitative PCR. Background Technology

[0002] Baked goods include bread, cakes, pastries, mooncakes, filled products, and ready-to-eat foods containing cream, meat floss, or other fillings. These foods typically contain a high proportion of starch, fat, sugar, and protein, and are at risk of contamination by foodborne pathogens such as Staphylococcus aureus and Bacillus cereus during raw material processing, post-baking cooling, filling, packaging, and storage.

[0003] Staphylococcus aureus can grow and produce enterotoxins under suitable conditions, while Bacillus cereus has the ability to form spores, some of which can survive after processing and germinate and proliferate under suitable conditions such as temperature and water activity. Therefore, rapid detection of these two pathogenic bacteria in baked goods is of great significance for food quality control and safety risk assessment.

[0004] While traditional culture methods can detect culturable bacteria, the detection cycle is usually long and requires multiple steps such as enrichment, isolation culture, and biochemical identification. Conventional PCR or quantitative real-time PCR has advantages such as high sensitivity, strong specificity, and fast detection speed, but it detects target DNA in the sample and cannot effectively distinguish between live and dead cells. When food has undergone heating, sterilization, or other treatments, DNA in dead cells may still exist for a considerable period. Conventional PCR will amplify this DNA, easily leading to false positives or overestimating the number of viable target bacteria.

[0005] Propidium azide bromide (PMA) is a photoreactive nucleic acid dye. PMA has difficulty penetrating intact membranes of living cells, but it can enter damaged dead cells and bind to DNA within them. Under specific wavelengths of light, PMA covalently cross-links with DNA, preventing the corresponding DNA from being effectively amplified by PCR. Therefore, combining PMA with quantitative real-time PCR can reduce the interference of dead cell DNA on detection results. However, baked goods matrices contain large amounts of starch, fat, protein, and pigments, which may affect the contact between PMA and dead cell DNA, the efficiency of PMA's photocrosslinking, bacterial DNA release, and PCR amplification. Furthermore, both Staphylococcus aureus and Bacillus cereus are Gram-positive bacteria, and their cell wall structures, lysis difficulties, and amplification efficiencies differ. Directly combining two singleton PCR systems can easily lead to problems such as primer-probe competition, fluorescence channel imbalance, and missed detection of low-abundance target bacteria.

[0006] Therefore, there is a need to establish a PMA-dual real-time PCR detection method suitable for the complex matrix of baked goods, which can effectively inhibit the amplification of DNA from dead cells and simultaneously detect Staphylococcus aureus and Bacillus cereus. Summary of the Invention

[0007] The purpose of this invention is to provide a method and kit for detecting foodborne pathogens based on PMA-dual fluorescence quantitative PCR.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for detecting foodborne pathogens based on PMA-dual fluorescence quantitative PCR, characterized in that the foodborne pathogens are Staphylococcus aureus and Bacillus cereus, and the method includes the following steps: (1) Sample pretreatment: The baked food sample to be tested was mixed with sterile buffered peptone water and homogenized. The resulting homogenate was centrifuged at low speed, the supernatant was collected and centrifuged at high speed, the bacterial precipitate was collected, and the bacterial precipitate was washed and resuspended with PBS buffer to obtain a bacterial suspension. (2) PMA treatment: Take the bacterial suspension, add propidium azidobromide to make the final concentration of propidium azidobromide 10 μmol / L, incubate in the dark and then irradiate with blue light to complete the first PMA treatment; after completing the first PMA treatment, centrifuge the treated bacterial suspension, discard the supernatant, resuspend the obtained bacterial precipitate with PBS buffer, add propidium azidobromide again to make the final concentration of propidium azidobromide 10 μmol / L, and complete the second PMA treatment by incubating in the dark and irradiating with blue light twice to obtain PMA-treated bacterial cells; (3) DNA extraction: PMA-treated bacteria were resuspended in an enzymatic hydrolysate containing lysozyme and lysostaphin for enzymatic hydrolysis. Proteinase K solution and DNA lysis buffer were added for lysis. The DNA template was obtained by centrifugation, silica gel column purification, washing and elution. (4) Dual fluorescence quantitative PCR detection: Using the DNA template as the amplification template, dual fluorescence quantitative PCR amplification was performed using the first primer and probe set targeting the nuc gene of Staphylococcus aureus and the second primer and probe set targeting the gyrB gene of Bacillus cereus. The probes of the first primer and probe set and the probes of the second primer and probe set were labeled with different reporter fluorescent groups, respectively. (5) Result determination: Based on the amplification curves and Ct values ​​of the two fluorescence channels, the presence of Staphylococcus aureus and Bacillus cereus in the tested baked food is determined, and the content of the two foodborne pathogens is determined according to the corresponding food matrix standard curve.

[0009] Preferably, in step (1), the mass-to-volume ratio of the baked food sample to be tested to the sterile buffered peptone water is 25 g: 225 mL; the homogenization time is 2 min; the centrifugal force of the low-speed centrifugation is 500×g, and the centrifugation time of the low-speed centrifugation is 2 min; the centrifugal force of the high-speed centrifugation is 10000×g, and the centrifugation time of the high-speed centrifugation is 5 min. The baked goods to be tested are selected from one of the following: bread, cake, cream-filled pastries, and mooncakes.

[0010] Preferably, in step (2), the volume of the bacterial suspension is 200 μL; the incubation time in the dark is 5 min; and the blue light irradiation conditions are irradiation under blue light with a wavelength of 465 nm for 10 min.

[0011] Preferably, in step (3), the volume of the enzymatic hydrolysate is 180 μL, the final concentration of lysozyme in the enzymatic hydrolysate is 20 mg / mL, the final concentration of lysostaphylococcal lysin is 100 U / mL, and the enzymatic hydrolysis condition is incubation at 37°C for 30 min. The amount of proteinase K solution added was 20 μL, the concentration of proteinase K solution was 20 mg / mL, the amount of DNA lysis buffer added was 200 μL, and the lysis conditions were incubation at 56°C for 20 min.

[0012] Preferably, the first primer-probe set includes: The upstream primer with the nucleotide sequence shown in SEQ ID NO.1: 5′-CAAAGCATCAAAAAGGTGTAGAGA-3′; The downstream primer with the nucleotide sequence shown in SEQ ID NO.2: 5′-TTCAATTTTCTTTGCATTTTCTACCA-3′; The probe has the nucleotide sequence shown in SEQ ID NO.3: 5′-TTTTCGTAATGCACTTGCTTCAGGACCA-3′; The second primer-probe set includes: an upstream primer with the nucleotide sequence shown in SEQ ID NO.4: 5′-GCCCTGGTATGTATATTGGATCTAC-3′; The downstream primer with the nucleotide sequence shown in SEQ ID NO.5: 5′-GGTCATAATAACTTCTACAGCAGGA-3′; The probe has the nucleotide sequence shown in SEQ ID NO. 6: 5′-CCATTTTTTCTTGTATAACCAACT-3′.

[0013] Preferably: the probe shown in SEQ ID NO.3 has a HEX fluorescent group labeled at its 5′ end and a BHQ1 quencher group labeled at its 3′ end; the probe shown in SEQ ID NO.6 has a FAM fluorescent group labeled at its 5′ end and an MGB modifying group attached to its 3′ end. In the dual real-time PCR reaction system, the final concentrations of the primers shown in SEQ ID NO.1 and SEQ ID NO.2 are 0.30 μmol / L, and the final concentration of the probe shown in SEQ ID NO.3 is 0.20 μmol / L; the final concentrations of the primers shown in SEQ ID NO.4 and SEQ ID NO.5 are 0.45 μmol / L, and the final concentration of the probe shown in SEQ ID NO.6 is 0.20 μmol / L. The total volume of the dual real-time PCR reaction was 25 μL, comprising: 12.50 μL of 2×qPCR Mix; 0.75 μL of the 10 μmol / L upstream primer shown in SEQ ID NO.1; 0.75 μL of the 10 μmol / L downstream primer shown in SEQ ID NO.2; 0.50 μL of the 10 μmol / L probe shown in SEQ ID NO.3; 1.125 μL of the 10 μmol / L upstream primer shown in SEQ ID NO.4; 1.125 μL of the 10 μmol / L downstream primer shown in SEQ ID NO.5; 0.50 μL of the 10 μmol / L probe shown in SEQ ID NO.6; 3.00 μL of DNA template; and 4.75 μL of nuclease-free water. The amplification program for the dual fluorescence quantitative PCR was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 45 s, for a total of 40 cycles, with FAM fluorescence signal and HEX fluorescence signal collected during the annealing and extension stage at 60℃.

[0014] Preferably, the result determination includes: When the HEX channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, Staphylococcus aureus is detected in the baked food being tested; when the FAM channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, Bacillus cereus is detected in the baked food being tested; when the Ct value of any channel is greater than 36 but not greater than 40, the detection result of that channel is considered suspicious, and the DNA template is tested repeatedly; when no typical S-shaped amplification curve appears in any channel within 40 amplification cycles, the target bacteria corresponding to that channel are not detected in the baked food being tested. When the Ct value is within the linear range of the corresponding food matrix standard curve, the content of the corresponding target bacteria is calculated based on the food matrix standard curve.

[0015] Secondly, the present invention provides a PMA-dual real-time PCR detection kit for detecting Staphylococcus aureus and Bacillus cereus in baked goods, comprising: propidium azidobromide; a first primer-probe set, comprising the upstream primer shown in SEQ ID NO.1, the downstream primer shown in SEQ ID NO.2, and the probe shown in SEQ ID NO.3; a second primer-probe set, comprising the upstream primer shown in SEQ ID NO.4, the downstream primer shown in SEQ ID NO.5, and the probe shown in SEQ ID NO.6; lysozyme; lysostaphin; proteinase K; DNA lysis buffer; probe-based real-time PCR premix; and positive control, negative control, and nuclease-free water.

[0016] Preferably, the propidium azide bromide is provided as a stock solution with a concentration of 1 mmol / L; the primers and probes shown in SEQ ID NO.1 to SEQ ID NO.6 are provided as solutions with a concentration of 10 μmol / L; the probe shown in SEQ ID NO.3 is labeled with a HEX fluorescent group at its 5′ end and a BHQ1 quencher group at its 3′ end; the probe shown in SEQ ID NO.6 is labeled with a FAM fluorescent group at its 5′ end and an MGB modifying group is attached to its 3′ end; the proteinase K is provided as a solution with a concentration of 20 mg / mL.

[0017] Preferably, the kit further includes sterile buffered peptone water, PBS buffer, enzymatic digestion solution, silica gel purification column, washing solution, and elution solution; The final concentration of lysozyme in the enzymatic hydrolysate is 20 mg / mL, and the final concentration of lysostaphylococcal lysin is 100 U / mL.

[0018] The beneficial effects of this invention are as follows: This invention removes debris, starch granules, and lipids from baked goods through differential centrifugation, while enriching the target bacteria and reducing interference from complex matrices on subsequent photocrosslinking, cell lysis, and PCR amplification. Two consecutive treatments with PMA at a final concentration of 10 μmol / L significantly inhibit the amplification of DNA from membrane-damaged dead bacteria, while having minimal impact on the amplification signal of intact cells, thus reducing false positives or overestimation of bacterial counts caused by residual dead bacterial DNA in conventional qPCR. A combined lysis using lysozyme, lysostaphin, and proteinase K improves the DNA release efficiency of two Gram-positive target bacteria. By balancing the concentrations of two sets of primers and probes (nuc and gyrB), Staphylococcus aureus and Bacillus cereus are simultaneously detected in the same reaction tube, reducing detection steps and reagent consumption. The established system is tested at 10... 2 ~10 7 It exhibits good linearity within the CFU / g range, with a detection limit of 10² CFU / g, good recovery rate and repeatability, and is suitable for rapid screening and semi-quantitative detection of two target bacteria in baked goods. Attached Figure Description

[0019] Figure 1 To optimize the amplification curve of Staphylococcus aureus under a dual qPCR system; Figure 2 To optimize the amplification curve of Bacillus cereus under a dual qPCR system; Figure 3 To optimize the standard curve for Staphylococcus aureus under a dual qPCR system; Figure 4 To optimize the standard curve of Bacillus cereus under a dual qPCR system; Figure 5 The amplification curve of Staphylococcus aureus in bread matrix; Figure 6 The amplification curve of Bacillus cereus in bread matrix; Figure 7 The standard curve for Staphylococcus aureus in bread matrix; Figure 8 This is the standard curve for Bacillus cereus in bread matrix. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It is important to note that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments of the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise.

[0021] Example 1 A method for detecting Staphylococcus aureus and Bacillus cereus in baked goods based on PMA-dual fluorescence quantitative PCR 1. Strains culture and preparation of bacterial culture (1) The standard strain of Staphylococcus aureus ATCC 25923 and the standard strain of Bacillus cereus ATCC14579 were selected as experimental strains. Staphylococcus aureus was inoculated in tryptic soy broth (TSB) and cultured at 37°C for 18 h to obtain the culture medium. Bacillus cereus was inoculated in nutrient broth and cultured at 30°C for 18 h to obtain the culture medium.

[0022] (2) After the culture was completed, the bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were washed twice with sterile PBS buffer, and the concentration of the bacterial culture was determined by plate counting. The bacterial culture was then serially diluted with PBS to: 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL.

[0023] 2. Sample processing of baked goods (1) Select bread, cake, cream-filled pastry and mooncake as the test objects. Aseptically weigh 25 g of sample, add 225 mL of sterile buffered peptone water, and process with a tapping homogenizer for 2 min to obtain food homogenized liquid.

[0024] (2) Take 10 mL of homogenized liquid and add it to a centrifuge tube. First, centrifuge at 500×g for 2 min to remove food debris, starch particles and large lipid particles.

[0025] (3) Take 8 mL of the supernatant, centrifuge at 10000×g for 5 min, and collect the bacterial precipitate.

[0026] (4) Resuspend the precipitate in 1 mL PBS and repeat the centrifugation and washing once.

[0027] 3. PMA processing (1) Prepare a 1 mmol / L PMA working solution and store it in the dark throughout the process. Take the bacterial precipitate obtained after sample pretreatment, resuspend it thoroughly with 198 μL of sterile PBS buffer, and then add 2 μL of 1 mmol / L PMA working solution to make the total volume of the reaction system 200 μL and the final concentration of PMA 10 μmol / L.

[0028] (2) Then place it in a 465 nm blue light irradiation device for 10 min, and repeat the above PMA treatment once to obtain the bacterial cell treatment solution.

[0029] 4. DNA extraction (1) The bacterial precipitate after PMA treatment was resuspended in 180 μL of enzymatic hydrolysate. The final concentration of lysozyme in the enzymatic hydrolysate was 20 mg / mL and the final concentration of lysostaphylococcal lysin was 100 U / mL. The mixture was incubated at 37°C for 30 min. During the incubation period, the mixture was gently inverted and mixed once every 10 min.

[0030] (2) After the enzymatic digestion is completed, add 20 μL of proteinase K solution with a concentration of 20 mg / mL to the reaction solution, then add 200 μL of 1× DNA lysis buffer, vortex mix for 15 s, and incubate at 56℃ for 20 min.

[0031] (3) After lysis, centrifuge at 12000×g for 2 min, take the supernatant, and purify the DNA using the silica gel column method. After washing, elute with 100 μL of elution buffer to obtain the DNA template.

[0032] 5. Establishment of a dual qPCR detection system (1) Target gene Staphylococcus aureus selects the nuc gene as its target gene, while Bacillus cereus selects the gyrB gene as its target gene.

[0033] (2) Primers and probes Staphylococcus aureus NUC detection system Upstream primer: 5'-CAAAGCATCAAAAAGGTGTAGAGA-3', SEQ ID NO.1; Downstream primer: 5'-TTCAATTTTCTTTGCATTTTCTACCA-3', SEQ ID NO.2; Probe: 5'-HEX-TTTTCGTAAATGCACTTGCTTCAGGACCA-BHQ1-3', SEQ ID NO.3; Bacillus cereus gyrB detection system Upstream primer: 5'-GCCCTGGTATGTATATTGGATCTAC-3', SEQ ID NO.4; Downstream primer: 5'-GGTCATAATAACTTCTACAGCAGGA-3', SEQ ID NO.5; Probe: 5'-FAM-CCATTTTTTCTTGTATACCAACT-MGB-3', SEQ ID NO.6.

[0034] (3) Dual qPCR reaction system Total reaction volume 25 μL:

[0035] (4) Amplification program Pre-denaturation at 95℃ for 3 min; 95℃ for 15 s; 60℃ for 45 s; 40 cycles, with FAM and HEX fluorescence signals collected at the 60℃ stage.

[0036] (5) Judgment method When the HEX channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, it is determined that Staphylococcus aureus has been detected in the baked food to be tested. When the FAM channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, it is determined that Bacillus cereus has been detected in the baked food to be tested. When the Ct value of any channel is greater than 36 and not greater than 40, the detection result of that channel is judged as suspicious, and the DNA template is tested repeatedly. If a typical S-shaped amplification curve does not appear in any channel within 40 amplification cycles, it is determined that the target bacteria corresponding to that channel are not detected in the baked food to be tested. When the Ct value is within the linear range of the corresponding food matrix standard curve, the content of the corresponding target bacteria is calculated based on the food matrix standard curve.

[0037] Example 2 PMA treatment condition optimization experiment 1. Preparation of bacterial suspension Staphylococcus aureus was inoculated into tryptic soy broth and cultured at 37°C and 180 r / min for 18 h; Bacillus cereus was inoculated into nutrient broth and cultured at 30°C and 180 r / min for 18 h.

[0038] Culture media were collected separately, centrifuged at 8000×g for 5 min to collect bacterial cells, washed twice with sterile PBS, and then resuspended in PBS. The bacterial concentration was determined by plate count method, and the viable cell concentration of the two bacterial suspensions was adjusted to 1×10^6 CFU / mL.

[0039] Each bacterial suspension was further divided into live and dead bacterial suspensions. Live bacterial suspensions were not inactivated; dead bacterial suspensions were treated in a 95°C water bath for 15 min and immediately cooled in an ice bath. 100 μL of each inactivated bacterial suspension was spread onto the corresponding culture medium. Staphylococcus aureus was incubated at 37°C for 48 h, and Bacillus cereus was incubated at 30°C for 48 h. No colony growth was observed after incubation, confirming complete bacterial inactivation.

[0040] 2. Experimental Grouping 200 μL of live bacterial suspension and 200 μL of dead bacterial suspension were respectively subjected to PMA treatment, and the settings for each group are as follows.

[0041] Table 1. Differences in treatment methods among groups

[0042] In Group A1, an equal volume of solvent as the PMA solution was added as a blank control, and the same light-protected incubation and blue light irradiation conditions were used as the other groups. Group A5 was a double-treatment group, with the final concentration of PMA in each treatment being 10 μmol / L, rather than adding 20 μmol / L PMA at once.

[0043] 3. PMA processing methods The experiment was conducted using 1 mmol / L PMA working solution. In group A2, PMA was added to the bacterial suspension to make the final PMA concentration 5 μmol / L; in groups A3 and A5, the final PMA concentration was 10 μmol / L after the first treatment; and in group A4, the final PMA concentration was 20 μmol / L.

[0044] After adding PMA, immediately vortex to mix for 5 seconds, then incubate at 20–25°C in the dark for 5 minutes. During incubation in the dark, gently invert the tube every 1 minute to ensure full contact between the PMA and the dead bacteria. After incubation, place the reaction tube in a blue light photolysis device and irradiate at 465±10 nm for 10 minutes. Maintain a distance of 8–10 cm between the reaction tube and the light source, rotate the reaction tube every 2 minutes during irradiation, and control the sample temperature to not exceed 25°C.

[0045] Groups A2, A3, and A4 underwent direct cell recovery after one light exposure. Group A5, after its first light exposure, was centrifuged at 10000×g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 198 μL of PBS. Then, 2 μL of 1 mmol / L PMA working solution was added to ensure a final PMA concentration of 10 μmol / L for the second treatment. The pellet was then incubated in the dark for 5 min and irradiated under 465±10 nm blue light for 10 min.

[0046] After all treatments were completed, the bacterial cells were collected by centrifugation at 10000×g for 5 min, the supernatant was discarded, and the bacterial cells were washed once with 200 μL PBS to remove free PMA.

[0047] 4. DNA extraction and dual qPCR detection DNA extraction and dual qPCR detection were performed as in Example 1. After detection, the Ct value of viable bacteria, the Ct offset of viable bacteria (Ct viable bacteria, PMA treatment - Ct viable bacteria, no PMA treatment), the Ct value of dead bacteria, the ΔCt of dead bacteria (Ct dead bacteria, PMA treatment - Ct dead bacteria, no PMA treatment), and the DNA amplification inhibition rate of dead bacteria ([1 - 2^(-ΔCt)] × 100%) were calculated.

[0048] The results are shown in Tables 2 and 3.

[0049] Table 2. Detection results of Staphylococcus aureus under different PMA treatment conditions.

[0050] Table 2 shows that the Ct values ​​of dead and live bacteria in group A1 were close, making it difficult for conventional qPCR to distinguish between them. 5 μmol / L PMA only increased the ΔCt of dead bacteria to 5.6, indicating insufficient inhibition. A single treatment with 10 μmol / L increased the ΔCt of dead bacteria to 13.4, achieving an inhibition rate of 99.9907%, while the Ct of live bacteria only shifted by 0.6. Although 20 μmol / L improved the inhibition of dead bacteria, the Ct of live bacteria shifted by 1.4. Two consecutive treatments with 10 μmol / L achieved a ΔCt of 14.5 for dead bacteria, achieving an inhibition rate of 99.9957%, while the Ct of live bacteria only shifted by 0.5, demonstrating the best overall effect.

[0051] Table 3. Detection results of Bacillus cereus under different PMA treatment conditions

[0052] Table 3 shows that even without PMA treatment, dead Bacillus cereus bacteria still significantly amplified, indicating that conventional qPCR is easily interfered with by dead bacterial DNA. 5 μmol / L PMA was insufficient for inhibition; a single treatment with 10 μmol / L resulted in a ΔCt of 12.8 for dead bacteria, achieving an inhibition rate of 99.9860%, while the Ct of viable bacteria shifted by only 0.6. Although 20 μmol / L enhanced inhibition, the Ct of viable bacteria shifted by 1.5. Two treatments with 10 μmol / L achieved a ΔCt of 13.9 for dead bacteria, an inhibition rate of 99.9935%, and a Ct shift of only 0.6 for viable bacteria, demonstrating the best overall effect.

[0053] 5. Further validation under high background dead bacteria conditions To further compare the detection capabilities of groups A3 and A5 in samples with high background dead bacteria, 1×10^4 CFU / mL of live bacteria and 1×10^7 CFU / mL of dead bacteria were mixed to make the number of dead bacteria approximately 1000 times that of live bacteria. The mixed samples were detected using conventional qPCR, a single treatment with 10 μmol / L PMA for group A3, and two treatments with 10 μmol / L PMA for group A5, and the number of live bacteria was calculated based on the corresponding standard curves. The results are shown in Table 4.

[0054] Table 4. Detection results of different treatment methods under high background dead bacteria conditions.

[0055] The results showed that, with the number of dead bacteria being 1000 times that of live bacteria, untreated qPCR overestimated the number of the two target bacteria by 2.83 log and 2.91 log, respectively. After a single treatment with 10 μmol / L PMA, the detection bias was reduced, but still reached 0.68 log and 0.74 log, respectively.

[0056] After two treatments with 10 μmol / L PMA, the detection biases for Staphylococcus aureus and Bacillus cereus decreased to 0.16 log and 0.19 log, respectively, both less than 0.5 log, indicating that the two-time treatment has better dead bacterial DNA exclusion effect and quantification accuracy in the context of high dead bacteria.

[0057] Example 3 Optimization of primer and probe concentrations in dual qPCR system (1) The strain treatment, PMA treatment and DNA extraction steps are the same as in Example 1.

[0058] (2) The concentrations of Staphylococcus aureus nuc primer, nuc probe, Bacillus cereus gyrB primer and gyrB probe were used as the factors to be investigated. Each factor was set with three levels, as shown in Table 5.

[0059] Table 5 Factors and levels in the double qPCR orthogonal experiment

[0060] (3) Nine groups of reactions were set up according to the L9 (3^4) orthogonal array, as shown in Table 6.

[0061] Table 6 L9 (3^4) Orthogonal Experimental Combinations

[0062] Each group has 3 independent reactions, and each reaction has 3 technical duplicate wells.

[0063] (4) The total volume of the dual qPCR reaction is 25 μL, including: 12.5 μL of 2× probe method fluorescent quantitative PCR premix; different concentrations of nuc upstream and downstream primers; different concentrations of nuc probe; different concentrations of gyrB upstream and downstream primers; different concentrations of gyrB probe; 3.0 μL of mixed DNA template; and nuclease-free water to make up to 25 μL.

[0064] Except for the primer and probe concentrations, which were adjusted according to Tables 5 and 6, the other reaction components remained the same.

[0065] Each batch of reactions simultaneously included: a Staphylococcus aureus singlet qPCR control; a Bacillus cereus singlet qPCR control; a dual-target positive control; and a template-free negative control.

[0066] (5) The amplification procedure is as follows: 1. Pre-denaturation at 95℃ for 3 min; 2. Denaturation at 95℃ for 15 s; 3. Annealing and extension at 60℃ for 45 s, and collect FAM and HEX fluorescence signals; 4. Repeat steps 2 and 3 for 40 cycles.

[0067] The amplification signal of the gyrB gene in Bacillus cereus was collected through the FAM channel, and the amplification signal of the nuc gene in Staphylococcus aureus was collected through the HEX channel.

[0068] (6) Results of orthogonal experiments Nine sets of primer-probe concentration combinations were subjected to duplex qPCR detection, with single qPCR using the same primer and probe concentrations as controls. Each set included three independent reactions, and each independent reaction had three technical replicates.

[0069] The Ct values ​​of each duplex qPCR group, the Ct offset relative to singlet qPCR, the Ct difference between the two channels, the standard deviation of the technique replicates, and the overall loss value L (L=|ΔCt-nuc|+|ΔCt-gyrB|+the Ct difference between the two channels+SD-nuc+SD-gyrB) are shown in Table 7.

[0070] Table 7. Results of dual qPCR detection with different primer-probe concentration combinations

[0071] As shown in Table 7, no significant nonspecific amplification was observed in any of the experimental groups. The comprehensive loss values ​​of Experiments 2, 4, and 5 were 0.80, 0.94, and 0.90, respectively, all below 1.0, indicating that the above combinations have good dual amplification performance.

[0072] In Experiment 2, the Ct values ​​for the nuc and gyrB channels were 26.92±0.19 and 27.06±0.21, respectively, with Ct shifts of only 0.12 and 0.14 compared to single qPCR. The Ct difference between the two channels was only 0.14, and the overall loss value was the lowest at 0.80, indicating that it performed best in terms of amplification efficiency, channel balance, and reproducibility. Experiment 1 had the highest overall loss value of 2.10, mainly due to the large Ct shifts and Ct difference between the two channels. In Experiment 7, the Ct shift of the nuc channel reached 0.55, leading to an increased overall loss value of 1.56. Overall, different primer-probe concentration combinations have a significant impact on channel balance and amplification stability in the dual qPCR system, and further range analysis is needed to determine the optimal levels of each factor.

[0073] (7) Range analysis of orthogonal experiments Based on the comprehensive scores of each group, the average score k and range R of each factor at different levels were calculated, and the results are shown in Table 8.

[0074] Table 8. Results of Range Analysis of Orthogonal Experiments

[0075] Range analysis results showed that the influence of each factor on the overall performance of the dual qPCR system was as follows: The order of influence is C > B > A > D, meaning the gyrB primer concentration has the greatest impact, followed by the nuc probe concentration, nuc primer concentration, and gyrB probe concentration. Specifically, the range R for factor C is 0.593, indicating that the gyrB primer concentration has the most significant impact on the amplification balance of the two targets in the dual-mode system. The ranges for factors B and A are 0.533 and 0.400, respectively, indicating that the nuc probe and nuc primer concentrations also have a significant impact on the overall system performance. The range for factor D is only 0.173, indicating that within the investigated concentration range, the gyrB probe concentration has a relatively small impact on the overall system performance.

[0076] Since a smaller overall loss value L indicates better overall system performance, and the lowest average loss values ​​for each factor all occur at level 2, the optimal levels for each factor are A2, B2, C2, and D2, respectively. Specifically: the final concentrations of the nuc upstream and downstream primers are each 0.30 μmol / L, and the final concentration of the nuc probe is 0.20 μmol / L; the final concentrations of the gyrB upstream and downstream primers are each 0.45 μmol / L, and the final concentration of the gyrB probe is 0.20 μmol / L.

[0077] (8) Optimal combination confirmation experiment Since the combination A2B2C2D2 is not directly included in the L9 orthogonal array, a confirmation experiment was further set up.

[0078] A dual qPCR system was prepared according to the A2B2C2D2 combination, and the two target DNAs were mixed in equal amounts as templates for detection. The preferred dual qPCR reaction system is shown in Table 9.

[0079] Table 9 Optimal Dual qPCR Reaction System

[0080] Confirmatory experimental results showed that the Ct value for the Staphylococcus aureus nucleus channel was 26.9 ± 0.18, and the Ct value for the Bacillus cereus gyrB channel was 27.1 ± 0.20, with a difference of 0.2 cycles between the two channels. Compared with the corresponding singlet qPCR, the Ct value for the nucleus channel was delayed by 0.23 cycles, and the Ct value for the gyrB channel was delayed by 0.27 cycles, both less than 1.0 cycle. No amplification was observed in the template-free control and the blank extraction control.

[0081] The results above demonstrate that the A2B2C2D2 combination can balance the amplification rate, channel balance, and stability of both detection channels.

[0082] (9) Verification of amplification efficiency of the optimized system Staphylococcus aureus and Bacillus cereus DNA were serially diluted 10-fold to ensure that the target bacterial equivalent in each reaction was 10. 2 10 3 10 4 10 5 10 6 and 10 7 CFU / mL.

[0083] Two DNAs of the same concentration gradient were mixed at a volume ratio of 1:1, and the amplification curve was detected using a preferred dual qPCR system, as shown in the figure. Figure 1 and Figure 2 As shown. A standard curve is constructed with the log10 value of the template concentration on the x-axis and the Ct value on the y-axis (the standard curve is shown in Figure 1). Figure 3 and Figure 4 As shown in the figure), the results are shown in Table 10.

[0084] Table 10 Gradient template detection results of the preferred dual qPCR system

[0085] from Figure 1-4 As shown in Table 10, the standard curve equation for the Staphylococcus aureus NUC channel is Ct = -3.432 log10N + 41.67; the correlation coefficient R0 2 The value was 0.996; the amplification efficiency was 95.6%.

[0086] The standard curve equation for the Bacillus cereus gyrB channel is Ct = -3.401 log10N + 41.89; the correlation coefficient R0 2 The value was 0.995; the amplification efficiency was 96.8%.

[0087] The correlation coefficients of the standard curves for both target bacteria were greater than 0.99, and the amplification efficiencies were both within the suitable range of 90%–110%. (In 10...) 2 ~10 7 Within the CFU / mL range, for every 10-fold decrease in template concentration, the corresponding Ct value increases by approximately 3.4 cycles, indicating that the optimized dual qPCR system has good linearity and amplification stability.

[0088] Based on the above results, the optimal combination was determined to be A2B2C2D2, that is, the final concentrations of the nuc upstream and downstream primers are each 0.30 μmol / L, the final concentration of the nuc probe is 0.20 μmol / L, the final concentrations of the gyrB upstream and downstream primers are each 0.45 μmol / L, and the final concentration of the gyrB probe is 0.20 μmol / L.

[0089] Example 4: Experiment on Artificial Contamination Detection of Food Samples To evaluate the linear range, detection sensitivity, semi-quantitative ability, and spiked recovery rate of the method of this invention in baked food matrices, negative... Bread samples were used as a representative food matrix for artificial contamination detection experiments of Staphylococcus aureus and Bacillus cereus.

[0090] 1. Screening of negative bread samples In this embodiment, plain white bread without fillings, butter, or meat products was selected as the experimental sample. The bread sample was pre-tested using both the culture method and the dual qPCR method of this invention. The culture method did not detect Staphylococcus aureus or Bacillus cereus. The dual qPCR method of this invention did not show typical amplification curves in either the FAM or HEX channels, confirming that the selected bread sample was a negative matrix.

[0091] 2. Target bacterial culture and bacterial suspension preparation Staphylococcus aureus was inoculated into tryptic soy broth and cultured at 37°C and 180 r / min for 18 h. Bacillus cereus was inoculated into nutrient broth and cultured at 30°C and 180 r / min for 18 h. After culture, bacterial cells were collected by centrifugation at 8000×g for 5 min, washed twice with sterile PBS, and then resuspended in PBS. The actual viable cell concentration of the two bacterial suspensions was determined by plate count method, and serial dilutions were performed 10-fold with sterile PBS.

[0092] 3. Aseptically weigh 25.0 g of the negative bread sample and place it in a sterile homogenizing bag.

[0093] Staphylococcus aureus and Bacillus cereus bacterial solutions were added to each bread sample simultaneously, so that the final concentrations of the two target bacteria in the sample were 1×10⁻⁶. 2 CFU / g; 1×10 3 CFU / g; 1×10 4 CFU / g; 1×10 5 CFU / g; 1×10 6 CFU / g; 1×10 7 CFU / g, with three independent samples for each concentration, and a negative control group of bread without the target bacteria was also set up.

[0094] After adding the bacterial solution, use a sterile glass rod or pipette to distribute the bacterial solution evenly in the bread sample, and let it stand at room temperature for 30 minutes to allow the target bacteria to fully contact the food matrix.

[0095] 4. Pretreatment of artificially contaminated samples (1) Add 225 mL of sterile buffered peptone water to 25.0 g of artificially contaminated bread sample, and homogenize it for 2 min at 250 times / min using a tapping homogenizer to obtain bread homogenized liquid.

[0096] (2) Take 10 mL of homogenized liquid and place it in a 15 mL sterile centrifuge tube. Centrifuge at 500×g for 2 min to remove larger bread crumbs and starch particles.

[0097] (3) Take 8 mL of the upper liquid and transfer it to a new centrifuge tube. Centrifuge at 10000×g and 4℃ for 5 min to collect the bacterial precipitate.

[0098] (4) Add 1.0 mL of PBS to the bacterial pellet, vortex for 30 s, centrifuge at 10000×g for 5 min, wash once, and finally resuspend the bacterial pellet in 198 μL of PBS.

[0099] 5. PMA processing The processing method is the same as in Example 1.

[0100] 6. DNA extraction The extraction method is the same as in Example 1.

[0101] 7. Dual qPCR detection The detection method is the same as in Example 1.

[0102] 8. Establishment of matrix standard curve The test results are shown in Table 11 and Figures 5-8 As shown.

[0103] Table 11. Results of dual qPCR detection of bread samples with different concentrations of artificial contamination.

[0104] From Table 11 and Figures 5-8 It can be seen that the standard curve equation for Staphylococcus aureus in bread matrix is ​​Ct = -3.360 log10N + 41.67; the correlation coefficient R0 2 The correlation coefficient was 0.993; the amplification efficiency was 98.4%. The standard curve equation for Bacillus cereus on bread matrix was Ct = -3.390 log10N + 42.08; the correlation coefficient R was 0.993. 2 The value was 0.991; the amplification efficiency was 97.2%.

[0105] The results show that at 1×10 2 ~1×10 7 Within the CFU / g range, the Ct values ​​of both target bacteria decreased regularly with increasing artificial contamination concentration, and the Ct value showed a good linear relationship with the log10 value of the target bacteria concentration.

[0106] 9. Spike recovery rate test The number of target bacteria in each artificially contaminated sample was converted according to the established matrix standard curve, and the spiked recovery rate was calculated according to the following formula: Spike recovery rate = (detected bacterial count / theoretical spiked bacterial count) × 100%. The test results are shown in Table 12.

[0107] Table 12 Spiked recovery results of artificially contaminated bread samples

[0108] Table 12 shows that the recoveries of Staphylococcus aureus at different spike concentrations ranged from 91.0% to 105.0%, and the recoveries of Bacillus cereus ranged from 88.0% to 106.0%. The recoveries of both target bacteria were within acceptable ranges, indicating that the starch, fat, and protein components in bread did not significantly interfere with the dual qPCR detection.

[0109] 10. Validation of Limit of Detection and Limit of Quantitation Three concentrations of 50, 100, and 200 CFU / g were further set near the initial detection limit. Twenty independent artificially contaminated samples were prepared for each concentration and detected according to the method of the present invention. The results are shown in Table 13.

[0110] Table 13 Low concentration detection results of the method of the present invention

[0111] As shown in Table 13, when the artificial contamination concentration was 50 CFU / g, there were partial missed detections for both target bacteria; when the artificial contamination concentration reached 100 CFU / g, the positive detection rate for both target bacteria reached 100%. At the 100 CFU / g level, the recoveries of Staphylococcus aureus and Bacillus cereus were 91.0% and 88.0%, respectively, and the standard deviation of the technical replicate Ct value did not exceed 0.50.

[0112] Therefore, under the bread matrix and sample pretreatment conditions of this embodiment, the detection limit and quantitation limit of both target bacteria were determined to be 1×10⁻⁶. 2 CFU / g.

[0113] 11. Repeatability testing Select 1×10 respectively 2 1×10 4 and 1×10 6 Three concentration levels of CFU / g were set up, with 6 independent samples at each level, and the same operator completed the testing on the same day.

[0114] Table 14 Repeatability test results of the method of the present invention

[0115] As shown in Table 14, the coefficient of variation of Ct values ​​at each concentration level is less than 1.5%, indicating that the method of the present invention has good repeatability.

[0116] 12. Negative control results The results showed that, after all pretreatments, PMA treatment, DNA extraction, and dual qPCR detection, negative bread samples without the target bacteria did not exhibit typical S-shaped amplification curves in either the FAM or HEX channels. No amplification was observed in the template-free control and the DNA extraction blank control, indicating that no significant target DNA contamination or non-specific amplification occurred during the experiment.

[0117] The results above show that, by artificially adding Staphylococcus aureus and Bacillus cereus to negative-negative bread as a matrix, the method of the present invention can achieve a yield of 1×10⁻⁶ bacteria. 2 ~1×10 7 Simultaneous detection of two target bacteria was performed within the CFU / g range.

[0118] Among them, the correlation coefficient R of the Staphylococcus aureus matrix standard curve 2 The correlation coefficient R of the Bacillus cereus matrix standard curve was 0.993, and the amplification efficiency was 98.4%; 2The amplification efficiency was 97.2%, with a value of 0.991. The recoveries of Staphylococcus aureus were 91.0%–105.0%, and the recoveries of Bacillus cereus were 88.0%–106.0%. The coefficients of variation of Ct values ​​at each concentration level were all less than 1.5%.

[0119] The results show that the present invention, through a dual qPCR reaction system involving food matrix purification, bacterial enrichment, double PMA treatment, compound lysis, and concentration balancing, can reduce the interference of bread matrix on the detection process and achieve rapid semi-quantitative detection of Staphylococcus aureus and Bacillus cereus in baked goods.

Claims

1. A method for detecting foodborne pathogens based on PMA-dual fluorescence quantitative PCR, characterized in that, The foodborne pathogens are Staphylococcus aureus and Bacillus cereus. The method is used to detect target bacteria in baked goods in the presence of a high background of dead bacteria. The method includes the following steps: (1) Sample pretreatment: The baked food sample to be tested was mixed with sterile buffered peptone water and homogenized. The resulting homogenate was centrifuged at low speed, the supernatant was collected and centrifuged at high speed, the bacterial precipitate was collected, and the bacterial precipitate was washed and resuspended with PBS buffer to obtain a bacterial suspension. (2) PMA treatment: Take the bacterial suspension, add propidium azidobromide to make the final concentration of propidium azidobromide 10 μmol / L, incubate in the dark and then irradiate with blue light to complete the first PMA treatment; after completing the first PMA treatment, centrifuge the treated bacterial suspension, discard the supernatant, resuspend the obtained bacterial precipitate with PBS buffer, add propidium azidobromide again to make the final concentration of propidium azidobromide 10 μmol / L, and complete the second PMA treatment by incubating in the dark and irradiating with blue light twice to obtain PMA-treated bacterial cells; (3) DNA extraction: PMA-treated bacteria were resuspended in an enzymatic hydrolysate containing lysozyme and lysostaphin for enzymatic hydrolysis. Proteinase K solution and DNA lysis buffer were added for lysis. The DNA template was obtained by centrifugation, silica gel column purification, washing and elution. (4) Dual fluorescence quantitative PCR detection: Using the DNA template as the amplification template, dual fluorescence quantitative PCR amplification was performed using the first primer and probe set targeting the nuc gene of Staphylococcus aureus and the second primer and probe set targeting the gyrB gene of Bacillus cereus. The probes of the first primer and probe set and the probes of the second primer and probe set were labeled with different reporter fluorescent groups, respectively. (5) Result determination: Based on the amplification curves and Ct values ​​of the two fluorescence channels, the presence of Staphylococcus aureus and Bacillus cereus in the tested baked food is determined, and the content of the two foodborne pathogens is determined according to the corresponding food matrix standard curve.

2. The method according to claim 1, characterized in that, The high dead bacteria background refers to the number of dead Staphylococcus aureus and / or Bacillus cereus in the test system being 1000 times the corresponding number of live bacteria; In step (1), the mass-to-volume ratio of the baked food sample to be tested to the sterile buffered peptone water is 25 g: 225 mL; the homogenization time is 2 min; the centrifugal force of the low-speed centrifugation is 500×g, and the centrifugation time of the low-speed centrifugation is 2 min; the centrifugal force of the high-speed centrifugation is 10000×g, and the centrifugation time of the high-speed centrifugation is 5 min. The baked goods to be tested are selected from one of the following: bread, cake, cream-filled pastries, and mooncakes.

3. The method according to claim 2, characterized in that, In step (2), the volume of the bacterial suspension is 200 μL; the incubation time in the dark is 5 min; and the blue light irradiation conditions are irradiation under blue light with a wavelength of 465 nm for 10 min.

4. The method according to claim 3, characterized in that, In step (3), the volume of the enzymatic hydrolysate is 180 μL, the final concentration of lysozyme in the enzymatic hydrolysate is 20 mg / mL, the final concentration of lysostaphylococcal lysin is 100 U / mL, and the enzymatic hydrolysis condition is incubation at 37°C for 30 min. The amount of proteinase K solution added was 20 μL, the concentration of proteinase K solution was 20 mg / mL, the amount of DNA lysis buffer added was 200 μL, and the lysis conditions were incubation at 56°C for 20 min.

5. The method according to claim 1, characterized in that, The first primer-probe set includes: The upstream primer with the nucleotide sequence shown in SEQ ID NO.1: 5′-CAAAGCATCAAAAAGGTGTAGAGA-3′; The downstream primer with the nucleotide sequence shown in SEQ ID NO.2: 5′-TTCAATTTTCTTTGCATTTTCTACCA-3′; The probe has the nucleotide sequence shown in SEQ ID NO.3: 5′-TTTTCGTAATGCACTTGCTTCAGGACCA-3′; The second primer-probe set includes: an upstream primer with the nucleotide sequence shown in SEQ ID NO.4: 5′-GCCCTGGTATGTATATTGGATCTAC-3′; The downstream primer with the nucleotide sequence shown in SEQ ID NO.5: 5′-GGTCATAATAACTTCTACAGCAGGA-3′; The probe has the nucleotide sequence shown in SEQ ID NO. 6: 5′-CCATTTTTTCTTGTATAACCAACT-3′.

6. The method according to claim 5, characterized in that: The probe shown in SEQ ID NO.3 has a HEX fluorescent group labeled at its 5′ end and a BHQ1 quencher group labeled at its 3′ end; the probe shown in SEQ ID NO.6 has a FAM fluorescent group labeled at its 5′ end and an MGB modifying group attached to its 3′ end. In the dual real-time PCR reaction system, the final concentrations of the primers shown in SEQ ID NO.1 and SEQ ID NO.2 are 0.30 μmol / L, and the final concentration of the probe shown in SEQ ID NO.3 is 0.20 μmol / L; the final concentrations of the primers shown in SEQ ID NO.4 and SEQ ID NO.5 are 0.45 μmol / L, and the final concentration of the probe shown in SEQ ID NO.6 is 0.20 μmol / L. The total volume of the dual real-time PCR reaction was 25 μL, comprising: 12.50 μL of 2×qPCR Mix; 0.75 μL of the 10 μmol / L upstream primer shown in SEQ ID NO.1; 0.75 μL of the 10 μmol / L downstream primer shown in SEQ ID NO.2; 0.50 μL of the 10 μmol / L probe shown in SEQ ID NO.3; 1.125 μL of the 10 μmol / L upstream primer shown in SEQ ID NO.4; 1.125 μL of the 10 μmol / L downstream primer shown in SEQ ID NO.5; 0.50 μL of the 10 μmol / L probe shown in SEQ ID NO.6; 3.00 μL of DNA template; and 4.75 μL of nuclease-free water. The amplification program for the dual fluorescence quantitative PCR was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 45 s, for a total of 40 cycles, with FAM fluorescence signal and HEX fluorescence signal collected during the annealing and extension stage at 60℃.

7. The method according to claim 6, characterized in that, The result determination includes: When the HEX channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, Staphylococcus aureus is detected in the baked food being tested; when the FAM channel shows a typical S-shaped amplification curve and the Ct value is not greater than 36, Bacillus cereus is detected in the baked food being tested; when the Ct value of any channel is greater than 36 but not greater than 40, the detection result of that channel is considered suspicious, and the DNA template is tested repeatedly; when no typical S-shaped amplification curve appears in any channel within 40 amplification cycles, the target bacteria corresponding to that channel are not detected in the baked food being tested. When the Ct value is within the linear range of the corresponding food matrix standard curve, the content of the corresponding target bacteria is calculated based on the food matrix standard curve.

8. A PMA-dual real-time PCR detection kit for detecting Staphylococcus aureus and Bacillus cereus in baked goods, characterized in that, include: Propidium azide bromide; a first primer-probe set, comprising the upstream primer shown in SEQ ID NO.1, the downstream primer shown in SEQ ID NO.2, and the probe shown in SEQ ID NO.3; The second primer and probe set includes the upstream primer shown in SEQ ID NO.4, the downstream primer shown in SEQ ID NO.5, and the probe shown in SEQ ID NO.6; lysozyme; lysostaphin; proteinase K; and DNA lysis buffer. Probe-based real-time PCR premix; as well as positive control, negative control and nuclease-free water.

9. The PMA-dual fluorescence quantitative PCR detection kit according to claim 8, characterized in that, The propidium azide bromide is provided as a stock solution with a concentration of 1 mmol / L; the primers and probes shown in SEQ ID NO.1 to SEQ ID NO.6 are provided as solutions with a concentration of 10 μmol / L; the probe shown in SEQ ID NO.3 is labeled with a HEX fluorescent group at its 5′ end and a BHQ1 quencher group at its 3′ end; The probe shown in SEQ ID NO.6 has a FAM fluorescent group labeled at its 5′ end and an MGB modified group attached to its 3′ end; the proteinase K is provided in a solution with a concentration of 20 mg / mL.

10. The PMA-dual fluorescence quantitative PCR detection kit according to claim 8 or 9, characterized in that, The kit also includes sterile buffered peptone water, PBS buffer, enzyme digestion solution, silica gel purification column, washing solution, and elution solution; The final concentration of lysozyme in the enzymatic hydrolysate is 20 mg / mL, and the final concentration of lysostaphylococcal lysin is 100 U / mL.

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