Eight-Foodborne-Pathogen Joint Detection Kit Based on Multiplex Probe Melting Curve Method

Abstract

The present invention provides a kit for joint detection of eight foodborne pathogenic bacteria based on the multiplex probe melting curve method, belonging to the technical field of microbial detection. The primer-probe Mix in the kit provided by the present invention includes primers shown in SEQ ID NO.1-16, probes shown in SEQ ID NO.17-24, and substrates shown in SEQ ID NO.25-32. The present invention uses 2 fluorescence channels and differentiates them through different Tm temperature peaks of substrate melting peaks in a single fluorescence channel. The present invention realizes the full detection of characteristic genes in 8 foodborne pathogenic bacteria (Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, Yersinia enterocolitica, Cronobacter sakazakii, Shigella) in a single-tube fluorescence quantitative PCR reaction, and has the advantages of simple operation, good specificity, high sensitivity and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to a kit for joint detection of eight foodborne pathogenic bacteria based on the multiplex probe melting curve method. Background Art

[0002] As the saying goes, "Food is the paramount necessity of the people, and food safety is of prime importance". In recent years, food safety incidents caused by foodborne pathogenic bacteria have been frequent, seriously threatening people's physical health and thus affecting national economy, people's livelihood and social stability. Foodborne pathogenic bacteria are accompanied by symptoms such as fever, nausea, vomiting and diarrhea, and the symptoms of different types of pathogenic bacteria may vary. Among many foodborne pathogenic bacteria, there are many zoonotic pathogenic bacteria, which cause human infection of foodborne pathogenic bacteria through the human digestive system with the food chain as the medium. The main ones are bacteria such as Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, Yersinia enterocolitica, Cronobacter sakazakii, and Shigella.

[0003] At present, the clinical diagnosis of foodborne pathogenic bacteria mainly relies on traditional culture isolation and biochemical identification methods. This method is cumbersome to operate, has a long cycle, high cost, and is prone to a large number of missed diagnoses. There is an urgent need for efficient, sensitive and specific detection methods in clinical tests. There are currently two main methods for detecting foodborne pathogenic bacteria: one is to use ordinary PCR gel electrophoresis for determination, which can achieve multiplex amplification in one tube, and judge the carriage of pathogenic bacteria through the size of the amplicon band. However, it is necessary to open the lid of the PCR product, which is easy to cause contamination and false positives; the other is the Taqman probe fluorescence quantitative PCR technology, which uses a completely closed-tube operation to semi-quantitatively detect the carriage of pathogenic bacteria through the amplification curve. However, due to the limitation of 4-5 channels of the fluorescence quantitative platform, the detection of eight foodborne pathogenic bacteria for a single sample requires at least 2 tubes for detection, which severely limits the detection throughput and increases the workload of public health services.

[0004] High resolution melting (HRM) detection technology is a technology that combines PCR amplification technology and melting curve analysis technology, and realizes gene sequence analysis by relying on a high-resolution temperature detection PCR instrument and a new type of saturated fluorescent dye. Due to the performance of being able to fix the DNA sequence of each bacterium to generate accurate Tm values through HRM, HRM can be applied to a variety of foodborne pathogenic bacteria. Because of its many advantages such as simple and efficient operation, fast speed, no need to open the tube after PCR, avoiding the chance of secondary contamination, and high detection sensitivity, this technology has been rapidly popularized and applied in clinical diagnosis and various gene detections in other biological related sciences.

[0005] Hu Shuangfang et al. detected 9 kinds of foodborne pathogenic bacteria by dividing primer pairs into two groups for HRM-real time PCR reaction. Group A included Campylobacter jejuni (Tm: 81.31°C ± 0.035), Shigella

[0006] (Tm: 86.88°C ± 0.082), Vibrio parahaemolyticus (Tm: 85.55°C ± 0.071), Staphylococcus aureus (Tm: 81.81°C ± 0.024), Listeria monocytogenes (Tm: 81.60°C ± 0.041). Group B included Cronobacter sakazakii (Tm: 82.70°C ± 0.068), Salmonella (Tm: 86.29°C ± 0.035), Escherichia coli O157: H7 (Tm: 84.08°C ± 0.091), Vibrio cholerae (Tm: 86.58°C ± 0.041). Among them, the Tm values of Campylobacter jejuni, Staphylococcus aureus, and Listeria monocytogenes differed by about 0.3°C, with very small differences and being difficult to distinguish. Moreover, Campylobacter jejuni, Staphylococcus aureus, and Listeria monocytogenes could not be detected simultaneously with Salmonella, and two reaction tubes were required for detection, making the detection cumbersome. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the present invention provides a kit for joint detection of eight foodborne pathogenic bacteria based on the multiplex probe melting curve method. The primer-probe Mix in the kit provided by the present invention includes primers shown in SEQ ID NO.1-16, probes shown in SEQ ID NO.17-24, and substrates shown in SEQ ID NO.25-32. The present invention uses 2 fluorescence channels and differentiates through different Tm temperature peaks of substrate melting peaks in a single fluorescence channel. The present invention realizes the detection of characteristic genes of 8 foodborne pathogenic bacteria (Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio spp., Yersinia enterocolitica, Cronobacter sakazakii, Shigella) in a single-tube fluorescence quantitative PCR reaction, and has advantages such as simple operation, good specificity, high sensitivity, and low cost.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a kit for joint detection of eight foodborne pathogenic bacteria based on the multiplex probe melting curve method, and the eight foodborne pathogenic bacteria are Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio spp., Yersinia enterocolitica, Cronobacter sakazakii, Shigella.

[0010] Specifically, the kit includes primer-probe Mix.

[0011] More specifically, the primer-probe Mix includes the primers shown in SEQ ID NO.1-16.

[0012] Preferably, the nucleotide sequence of the upstream primer of Cronobacter sakazakii is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the upstream primer of Salmonella is as shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.4;

[0014] The nucleotide sequence of the upstream primer of Staphylococcus aureus is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.6;

[0015] The nucleotide sequence of the upstream primer of Escherichia coli O157 is as shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8;

[0016] The nucleotide sequence of the upstream primer of Shigella is as shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.10;

[0017] The nucleotide sequence of the upstream primer of Listeria monocytogenes is as shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.12;

[0018] The nucleotide sequence of the upstream primer of Vibrio is as shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.14;

[0019] The nucleotide sequence of the upstream primer of Yersinia enterocolitica is as shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.16.

[0020] More specifically, the primer-probe Mix further includes the probes shown in SEQ ID NO.17-24, and the probes can be labeled with a fluorescent group or a quenching group at the 5' end or the 3' end.

[0021] Preferably, the probe of Cronobacter sakazakii has the nucleotide sequence as shown in SEQ ID NO.17;

[0022] The probe of Salmonella has the nucleotide sequence as shown in SEQ ID NO.18;

[0023] The probe for Staphylococcus aureus has the nucleotide sequence shown in SEQ ID NO. 19;

[0024] The probe for Escherichia coli O157 has the nucleotide sequence shown in SEQ ID NO. 20;

[0025] The probe for Shigella has the nucleotide sequence shown in SEQ ID NO. 21;

[0026] The probe for Listeria monocytogenes has the nucleotide sequence shown in SEQ ID NO. 22;

[0027] The probe for Vibrio has the nucleotide sequence shown in SEQ ID NO. 23;

[0028] The probe for Yersinia enterocolitica has the nucleotide sequence shown in SEQ ID NO. 24.

[0029] More specifically, the primer-probe Mix further includes substrates shown in SEQ ID NOs. 25 - 32, and the substrates are labeled with a quenching group at the 3'-end.

[0030] Preferably, the substrate for Cronobacter sakazakii has the nucleotide sequence shown in SEQ ID NO. 25;

[0031] The substrate for Salmonella has the nucleotide sequence shown in SEQ ID NO. 26;

[0032] The substrate for Staphylococcus aureus has the nucleotide sequence shown in SEQ ID NO. 27;

[0033] The substrate for Escherichia coli O157 has the nucleotide sequence shown in SEQ ID NO. 28;

[0034] The substrate for Shigella has the nucleotide sequence shown in SEQ ID NO. 29;

[0035] The substrate for Listeria monocytogenes has the nucleotide sequence shown in SEQ ID NO. 30;

[0036] The substrate for Vibrio has the nucleotide sequence shown in SEQ ID NO. 31;

[0037] The substrate for Yersinia enterocolitica has the nucleotide sequence shown in SEQ ID NO. 32.

[0038] Preferably, the fluorescent group includes but is not limited to one or more of FAM, VIC, TET, CAL Gold 540, JOE, HEX, TAMRA, ROX, CY3, and CY5.

[0039] More preferably, the fluorescent group is selected from FAM or VIC.

[0040] Preferably, the quenching group includes but is not limited to one or more of DABCYL, BHQ1, BHQ2, BHQ3, and ECLIPE.

[0041] More preferably, the quenching group is BHQ1.

[0042] Specifically, the kit differentiates different target genes by different melting peak positions of the double-stranded products with corresponding different Tm values through fluorescence quenching in a single channel in the melting curve.

[0043] Preferably, the Tm temperature peak values of the substrate melting peaks of the target genes are as follows:

[0044] In the FAM fluorescence channel, the Tm temperature peak value of Cronobacter sakazakii is 61.2 °C, the Tm temperature peak value of Salmonella is 69.5 °C, the Tm temperature peak value of Staphylococcus aureus is 75 °C, and the Tm temperature peak value of Escherichia coli O157 is 79.5 °C;

[0045] In the VIC fluorescence channel, the Tm temperature peak value of Shigella is 60.5 °C, the Tm temperature peak value of Listeria monocytogenes is 67.7 °C, the Tm temperature peak value of Vibrio is 75.3 °C, and the Tm temperature peak value of Yersinia enterocolitica is 79.9 °C.

[0046] Specifically, the kit further includes a fluorescence PCR reaction premix.

[0047] Preferably, the fluorescence PCR reaction premix contains a PCR buffer, MgSO4, dNTPs, and Taq enzyme.

[0048] Specifically, the kit further includes a positive control.

[0049] Preferably, the positive control is a mixed solution containing amplification sequences of 8 target genes.

[0050] Specifically, the kit further includes a negative control.

[0051] Preferably, the negative control is enzyme-free and sterile water.

[0052] Specifically, the PCR amplification reaction conditions of the multiplex probe melting curve method are carried out according to the following steps:

[0053] (1) Amplification stage: Pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 20 s, for 45 cycles.

[0054] (2) Melting curve stage: 60°C for 5 s; 35°C for 15 s, for 15 cycles; total denaturation at 95°C for 60 s, 53°C for 60 s, fluorescence signals were collected at a heating rate of 0.06°C / s from 53°C to 85°C.

[0055] (3) Instrument cooling stage: 25°C for 1 min.

[0056] On the other hand, the present invention provides the application of the above-mentioned kit in the joint detection of eight foodborne pathogenic bacteria.

[0057] On another aspect, the present invention provides a method for the joint detection of eight foodborne pathogenic bacteria, the method comprising using the kit according to any one of claims 1-8.

[0058] Specifically, the method comprises the following steps:

[0059] S1. Extract nucleic acid of the sample to be tested using a nucleic acid extraction kit;

[0060] S2. Take the fluorescence PCR reaction premix and primer-probe Mix to prepare the fluorescence PCR reaction liquid;

[0061] S3. Take the nucleic acid extracted in step S1, positive control, and negative control, and add them to the fluorescence PCR reaction liquid in step S2 respectively;

[0062] S4. Select an automatic fluorescence PCR detector for detection.

[0063] Specifically, the automatic fluorescence PCR detector is a multi-channel calibrated automatic fluorescence PCR detector containing FAM and VIC (HEX) detection channels.

[0064] The technical effects achieved by the present invention:

[0065] (1) High detection throughput: All genes related to eight foodborne pathogenic bacteria can be detected in a single tube, and the operation is simple.

[0066] (2) Good specificity: The whole process is a closed-tube operation in a single tube on a fluorescence quantitative platform, which can avoid aerosol contamination caused by opening the lid and avoid false positive results caused by contamination.

[0067] (3) High sensitivity: The present invention is a technical upgrade based on the Taqman probe method, with strong anti-interference ability, high detection sensitivity, and easy result interpretation. Description of the Drawings

[0068] Figure 1 Schematic diagram of the results of octuple detection of foodborne pathogenic bacteria positive plasmids.

[0069] Figure 2 Schematic diagram of the results of octuple detection of NTC.

[0070] Figure 3 Schematic diagram of the results of octuple detection of Salmonella samples.

[0071] Figure 4 Schematic diagram of the results of octuple detection of Staphylococcus aureus samples.

[0072] Figure 5 Schematic diagram of the results of octuple detection of Escherichia coli O157 samples.

[0073] Figure 6 Schematic diagram of the results of octuple detection of Listeria monocytogenes samples.

[0074] Figure 7 Schematic diagram of the results of octuple detection of Vibrio samples.

[0075] Figure 8 Schematic diagram of the results of octuple detection of Yersinia enterocolitica samples.

[0076] Figure 9 Schematic diagram of the results of octuple detection of Cronobacter sakazakii samples.

[0077] Figure 10 Schematic diagram of the results of octuple detection of Shigella samples. Detailed implementation manners

[0078] The present invention will be further clearly and completely described below through embodiments. The following embodiments are only a part of the embodiments of the present invention, and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are not specially stated, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0079] In the following embodiments, the target genes are Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, Yersinia enterocolitica, Cronobacter sakazakii, Shigella, the positive control is a mixed solution containing amplification sequences of 8 target genes, and the negative control is enzyme-free and sterile water.

[0080] Among them, the interpretation of the detection results is as follows:

[0081] Positive control interpretation: If there are melting peaks in both the FAM and VIC channels, corresponding to 8 targets, and the Tm values are within the reference range, it can be determined that the positive control quality control is qualified; otherwise, it is determined that the positive control quality control is unqualified, and the experimental results of this time are invalid.

[0082] Negative control interpretation: There are no melting peaks in the two fluorescence channels. If there is a melting peak in any fluorescence channel of the negative control, it indicates that the negative control quality control is unqualified, suggesting that there is nucleic acid contamination in the operating environment, and false positive results may occur in the synchronous detection samples.

[0083] Test sample interpretation: The foodborne pathogenic bacteria in the sample are determined by the combination of the melting points (Tm values) of the melting curves of the two channels of the detected sample. The corresponding channels and reference values of Tm for each pathogenic bacterium are shown in part of Table 6.

[0084] Operation method of the multiplex detection kit for eight foodborne pathogenic bacteria in Example 1

[0085] Primer-probe Mix is designed according to the specific sequences of different foodborne pathogenic bacteria to detect whether the sample contains Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio spp., Yersinia enterocolitica, Cronobacter sakazakii, and Shigella. The primer, probe, and substrate sequence information of each target gene is shown in Tables 1 - 3, and then the primer-probe Mix is prepared according to Table 4.

[0086] Table 1 Eight pairs of primers in the primer-probe Mix

[0087]

[0088] Table 2 Eight probes in the primer-probe Mix

[0089]

[0090] Table 3 Eight substrates in the primer-probe Mix

[0091]

[0092]

[0093] Table 4 Preparation table of the primer-probe Mix

[0094]

[0095]

[0096] The content of each component of the fluorescence PCR reaction premix of the present invention is shown in Table 5:

[0097] Table 5 Content of each component of the fluorescence PCR reaction premix

[0098] Component Volume (μL) PCR Buffer 2 <![CDATA[25mM MgSO4]]> 0.8 dNTPs (25 mM, A:C:G:T = 1:1:1:1) 0.1 Taq Enzyme 0.2 Nuclease-Free Water 9.9 Total Volume 13

[0099] 1. Reagent Preparation

[0100] Remove all reagents from the refrigerator and equilibrate them to room temperature. The standard configuration of the fluorescent PCR reaction solution is as follows: Take n×13 μL of the fluorescent PCR reaction premix and n×2 μL of the primer-probe Mix and add them to a 1.5 mL centrifuge tube (n is determined according to the requirements of the current experiment). Vortex for several seconds and centrifuge briefly. Then, aliquot 15 μL of the PCR reaction solution into each PCR reaction tube. It is best to use the prepared PCR reaction solution within 4 hours.

[0101] 2. Sample Extraction and Loading

[0102] All required samples are enrichment broths of food samples, including Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio spp., Yersinia enterocolitica, Cronobacter sakazakii, and Shigella spp. Extract nucleic acids using a conventional bacterial nucleic acid extraction kit. Use a micropipette to add the corresponding extracted nucleic acids to each well of the PCR reaction tube and immediately seal the tube lid tightly.

[0103] 3. PCR Amplification

[0104] Place the prepared PCR reaction tubes into the fluorescence quantitative PCR instrument. After verifying that the reaction program, fluorescence collection, and channel selection are correct, start the reaction program. Amplification stage: Pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 20 s (collect fluorescence), 45 cycles; melting curve stage: 60 °C for 5 s, 35 °C for 15 s, 15 cycles; total denaturation at 95 °C for 60 s, final extension at 58 °C for 60 s, 58 - 83 °C (continuously collect fluorescence, heating rate 0.06 °C / s); instrument cooling stage: 25 °C for 1 min. Collect fluorescence in a total of 2 fluorescence channels, FAM and VIC.

[0105] After the program runs to completion, remove the PCR reaction tubes (closed tubes) and place them into a self-sealing bag. Seal the bag tightly and handle them according to the source of pollution.

[0106] The experimental results show that the corresponding channels and Tm values for different foodborne pathogenic bacteria are as shown in Table 6.

[0107] Table 6 Reference Value Ranges for Eight Foodborne Pathogenic Bacteria

[0108]

[0109] Example 2 Determination of Positive Control Plasmid

[0110] Synthesize a positive control plasmid containing 8 pathogen target genes in Bio-Engineering (Shanghai) Co., Ltd., and serially dilute it into samples with copy numbers of 3.2×10 4 copies / mL, 8×10 3 copies / mL, 2×10 3 copies / mL, 5×10 2 copies / mL. Use the kit of the present invention to operate according to Example 1, and detect templates with different dilution degrees respectively. The specific detection results are shown in Table 7, and it is obtained that the lowest detection limits of all 8 targets can reach 5×10 2 copies / mL.

[0111] Table 7 Test results of the lowest detection limits of eight foodborne pathogenic bacteria

[0112]

[0113] Note: + indicates that the sample is diluted to this gradient and repeatedly detected 20 times, and all 20 detection results are positive; - indicates that the sample is diluted to this gradient and repeatedly detected 20 times, and at least one detection is negative in the 20 detections.

[0114] Meanwhile, the detection results for 8 positive plasmids are as Figure 1 shown, and the corresponding NTC detection results are as Figure 2 shown. It is found through the results that the positions of the characteristic peaks of the melting curves are correct, and the characteristic melting peaks of different genes in the same fluorescence channel can be clearly distinguished, and the effect of detecting 8 targets with 2 fluorescence channels can be achieved. Therefore, the kit described in the present invention can be used for the detection of eight foodborne pathogenic bacteria.

[0115] Example 3 Determination of genomic DNA of 8 foodborne pathogenic bacteria

[0116] For positive samples of Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, Yersinia enterocolitica, Cronobacter sakazakii, and Shigella, extract the genomic DNA of 8 foodborne pathogenic bacteria. Serial dilute the 8 nucleic acids to be detected into samples equivalent to 3.2×10 4 copies / mL, 8×10 3 copies / mL, 2×10 3 copies / mL, 5×10 2 copies / mL.

[0117] Use the kit of the present invention to detect templates with 7 types of serial dilutions respectively according to the operation in Example 1, and the positive detection results are as Figure 3 , Figure 4 , Figure 5, Figure 6 , Figure 7 , Figure 8 , Figure 9 and Figure 10 , the results showed that Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio spp., Yersinia enterocolitica, Cronobacter sakazakii, and Shigella were all positive. The minimum detection limit results of the 8 target pathogens are shown in Table 8, and the detection results showed that the minimum detection limits of the 8 types of targets could all reach 5*10 2 copies / mL.

[0118] Table 8 Minimum detection limit test results of eight foodborne pathogenic bacteria

[0119]

[0120] Note: + indicates that the sample was diluted to this gradient and repeatedly detected 20 times, and all 20 detection results were positive; - indicates that the sample was diluted to this gradient and repeatedly detected 20 times, and at least one of the 20 detections was negative.

[0121] Comparative Example 1

[0122] According to the primers, probes, and substrates of Salmonella described in Patent CN116426619A, the primer-probe Mix in the kit of the present invention was replaced. Except for this, the operation steps were the same as those in Example 1 of the present invention. The primer, probe, and substrate information of each target gene in Comparative Example 1 is shown in Tables 9-11.

[0123] Table 9 Primers of Comparative Example 1

[0124]

[0125]

[0126] Table 10 Probes of Comparative Example 1

[0127]

[0128] Table 11 Substrates of Comparative Example 1

[0129]

[0130]

[0131] The experimental results found that the corresponding channels and Tm values of different foodborne pathogenic bacteria are shown in Table 12.

[0132] Table 12 Reference value ranges of eight foodborne pathogenic bacteria

[0133]

[0134] The measurement results show that in the FAM fluorescence channel, the difference in the peak Tm temperature between Cronobacter sakazakii and Salmonella is very small. In the VIC fluorescence channel, the difference in the peak Tm temperature between Shigella and Listeria monocytogenes is very small, and the difference in the peak Tm temperature between Vibrio and Yersinia enterocolitica is very small. The above results indicate that it is difficult to distinguish eight foodborne pathogenic bacteria with the kit prepared in Comparative Example 1.

[0135] Comparative Example 2

[0136] According to the primers, probes, and substrates of Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, and Yersinia enterocolitica described in Patent CN116426619A, the primer-probe Mix in the kit of the present invention was replaced. Except for this, the operation steps were the same as those in Example 1 of the present invention. The primer, probe, and substrate sequence information of each target gene in Comparative Example 2 can be seen in Tables 13 - 15.

[0137] Table 13 Primers of Comparative Example 2

[0138]

[0139]

[0140] Table 14 Probes of Comparative Example 1

[0141]

[0142] Table 15 Substrates of Comparative Example 1

[0143]

[0144] The experimental results show that the corresponding channels and Tm values of different foodborne pathogenic bacteria are as shown in Table 16.

[0145] Table 16 Reference value ranges of eight foodborne pathogenic bacteria

[0146]

[0147] The measurement results show that in the FAM fluorescence channel, the difference in the peak Tm temperature between Salmonella and Staphylococcus aureus is very small and difficult to distinguish. In the VIC fluorescence channel, it is impossible to distinguish between Listeria monocytogenes and Vibrio. The above results indicate that it is difficult to distinguish eight foodborne pathogenic bacteria with the kit prepared in Comparative Example 2.

[0148] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A kit for the joint detection of eight foodborne pathogens based on a multiple probe melting curve method, characterized in that: The kit includes a primer-probe Mix, which is composed of primers shown in SEQ ID NOs.1-16, probes shown in SEQ ID NOs.17-24, and substrates shown in SEQ ID NOs.25-32; The eight foodborne pathogens are Salmonella, Staphylococcus aureus, Escherichia coli O157, Listeria monocytogenes, Vibrio, Yersinia enterocolitica, Cronobacter sakazakii, and Shigella; The probe described in SEQ ID NO.18 in the probe is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end; the substrate is labeled with a quenching group at the 3' end; the fluorescent group is FAM; and the quenching group is BHQ1.

2. The kit according to claim 1, characterized in that The kit distinguishes different pathogenic bacteria in a single channel through the different melting peak positions of the corresponding double-stranded products with different Tm values ​​in the melting curve.

3. The kit according to claim 2, characterized in that In the FAM fluorescence channel, the Tm temperature peak of Cronobacter sakazakii was 61.2°C, the Tm temperature peak of Salmonella was 69.5°C, the Tm temperature peak of Staphylococcus aureus was 75°C, and the Tm temperature peak of Escherichia coli O157 was 79.5°C; In the VIC fluorescence channel, the Tm temperature peak of Shigella was 60.5°C, the Tm temperature peak of Listeria monocytogenes was 67.7°C, the Tm temperature peak of Vibrio was 75.3°C, and the Tm temperature peak of Yersinia enterocolitica was 79.9°C.

4. The kit according to claim 1, characterized in that The kit also includes a fluorescent PCR reaction premix, a positive control substance, and a negative control substance.

5. The kit according to claim 4, characterized in that: The fluorescent PCR reaction premix contains PCR buffer, MgSO4, dNTPs and Taq enzyme; the positive control is a mixed solution containing 8 target gene amplification sequences; and the negative control is enzyme-free sterile water.

6. The kit according to claim 1, characterized in that The PCR amplification reaction conditions of the multiple probe melting curve method are as follows: (1) Amplification stage: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 20 s, for 45 cycles; (2) Melting curve stage: 60°C for 5 s; 35°C for 15 s, for 15 cycles; total denaturation at 95°C for 60 s, 53°C for 60 s, and fluorescence signal collection from 53°C to 85°C at a heating rate of 0.06°C / s; (3) Instrument cooling stage: 25℃ for 1 min.

7. Use of the kit according to any one of claims 1 to 6 in the joint detection of eight foodborne pathogens, characterized in that: The application described is for non-diagnostic purposes.

8. A method for joint detection of eight foodborne pathogens, characterized in that: The method comprises using the kit according to any one of claims 1 to 6, and the method is a method for non-diagnostic purposes, comprising the following steps: S1. Use a nucleic acid extraction kit to extract nucleic acid from the sample to be tested; S2, taking fluorescent PCR reaction premix and primer probe Mix, and preparing fluorescent PCR reaction liquid; S3, taking the nucleic acid of the sample to be tested, the positive control substance and the negative control substance extracted in step S1, and adding them into the fluorescent PCR reaction liquid in step S2 respectively; S4. Use a fully automatic fluorescent PCR detector for testing.

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