Primer probe composition and kit for simultaneously detecting 17 pathogenic microorganisms in food
By using fluorescence quantitative PCR method and specific primer probe composition in food pathogenic microorganism detection technology, combined with enhanced PCR buffer and optimized PCR amplification program, the problem that the existing technology cannot detect multiple pathogenic microorganisms at the same time is solved, and efficient and accurate multi-target detection effect is achieved.
Patent Information
- Application Number
- CN202510414042.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-05-13
AI Technical Summary
The existing pathogenic microbial detection technology in food cannot detect multiple pathogenic microbials quickly and accurately at the same time, and there are problems such as inaccurate detection results and long time.
The primer probe composition based on fluorescence quantitative PCR method was used to design specific primers and probes for detecting pathogenic microorganisms in 17 foods, combined with enhanced PCR buffer and optimized PCR amplification program to achieve multi-target detection of quadruple fluorescence quantitative PCR.
It has achieved efficient and accurate detection of pathogenic microorganisms in 17 types of food, simplified the operation process, shortened the detection time, and improved the reliability and repetition of the detection results.
Smart Images
Figure CN119979776A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of food detection and relates to a primer-probe combination and a kit for simultaneously detecting pathogenic microorganisms in 17 kinds of food. Background Art
[0002] Pathogenic microorganisms in food are common pathogens that cause food poisoning. These pathogenic microorganisms can cause serious food poisoning incidents and even threaten life safety. my country's food supervision and random inspections and foodborne disease monitoring data also show that the reporting rate of foodborne diseases has generally shown an upward trend in recent years. Pathogenic microorganisms in food have become one of the main causes of food safety hazards. Therefore, the development of a rapid and accurate detection method is of great significance for the prevention and control of foodborne diseases.
[0003] At present, the commonly used detection technologies for pathogenic microorganisms in food are traditional culture methods, including ELISA technology, biosensor technology, biochip detection methods and PCR technology. The traditional culture method is easily interfered by other microorganisms in the sample during the detection process, resulting in inaccurate test results. ELISA technology is based on the principle of specific binding of antigen and antibody. It is simple and fast to operate, but its sensitivity is insufficient and it is easy to cause false negative results. Although biosensor technology has the advantages of high sensitivity and rapid response, its stability and repeatability are poor, and the cost is high. Although the biochip detection method can detect multiple pathogenic microorganisms at the same time, its preparation process is complicated and has high technical requirements for operators. Conventional PCR method identifies single bacterial pathogens in food by detecting specific DNA sequences. It has the advantages of strong specificity and low equipment cost, but its amplification product needs to be analyzed by agarose gel electrophoresis, which has limited sensitivity and resolution and takes a long time. Unlike conventional PCR method, real-time fluorescence quantitative PCR method does not require post-amplification treatment, but uses fluorescent dyes or probes to monitor PCR products in real time, which has the characteristics of rapid response, high specificity and high sensitivity.
[0004] Locked nucleic acids (LNA) are a new class of nucleic acid analogs that contain a 2'-O, 4'-C methylene bridge. This bridge is locked in the 3'-endo conformation, restricting the flexibility of the furanose ring and locking the structure into a rigid bicyclic form. When LNA is incorporated into qPCR probes, it offers several advantages over native DNA bases, including: greater thermal stability and hybridization specificity, more accurate gene quantification and allele discrimination, and easier and more flexible probe design for problematic target sequences. With LNA qPCR probes, the selective placement of LNA bases facilitates the optimal design of highly specific, shorter qPCR probes that perform well even with only 13 to 20 bases. Summary of the invention
[0005] The purpose of the present invention is to provide a primer-probe combination and a kit for simultaneously detecting pathogenic microorganisms in 17 kinds of food based on fluorescent quantitative PCR method. The primer-probe combination has strong specificity, simple operation, convenience and rapidity, reliable results and good repeatability.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A set of primer-probe combinations for simultaneously detecting pathogenic microorganisms in 17 kinds of food, including primers and probes for detecting Salmonella, Staphylococcus aureus, Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, Vibrio cholerae, Campylobacter jejuni, Clostridium botulinum, Proteus, group A rotavirus, Norovirus GⅠ, Norovirus GⅡ GⅡ), primer and probe combinations for Astrovirus, Sapovirus and Enteric Adenovirus;
[0008] in:
[0009] A forward primer Sal-F, a reverse primer Sal-R and a probe Sal-P for specifically detecting the Salmonella invA gene, wherein the primer sequences are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.3;
[0010] A forward primer Cpe-F, a reverse primer Cpe-R and a probe Cpe-P for specifically detecting the plc gene of Clostridium perfringens, wherein the primer sequences are shown in SEQ ID NO.4-5, and the probe sequence is shown in SEQ ID NO.6;
[0011] A forward primer Esc-F, a reverse primer Esc-R and a probe Esc-P for specifically detecting the rfbE gene of Escherichia coli O157, wherein the primer sequences are shown in SEQ ID NO.7-8, and the probe sequence is shown in SEQ ID NO.9;
[0012] A forward primer Vpa-F, a reverse primer Vpa-R and a probe Vpa-P for specifically detecting the toxS gene of Vibrio parahaemolyticus, wherein the primer sequences are shown in SEQ ID NO.10-11, and the probe sequence is shown in SEQ ID NO.12;
[0013] A forward primer Sau-F, a reverse primer Sau-R and a probe Sau-P for specifically detecting the nuc gene of Staphylococcus aureus, wherein the primer sequences are shown in SEQ ID NO.13-14, and the probe sequence is shown in SEQ ID NO.15;
[0014] A forward primer Vch-F, a reverse primer Vch-R and a probe Vch-P for specifically detecting the hlyA gene of Vibrio cholerae, wherein the primer sequences are shown in SEQ ID NO.16-17, and the probe sequence is shown in SEQ ID NO.18;
[0015] A forward primer Bac-F, a reverse primer Bac-R and a probe Bac-P for specifically detecting the cerB gene of Bacillus cereus, wherein the primer sequences are shown in SEQ ID NO.19-20, and the probe sequence is shown in SEQ ID NO.21;
[0016] A forward primer Lmo-F, a reverse primer Lmo-R and a probe Lmo-P for specifically detecting the actA gene of Listeria monocytogenes, wherein the primer sequences are shown in SEQ ID NO.22-23, and the probe sequence is shown in SEQ ID NO.24;
[0017] A forward primer Cje-F, a reverse primer Cje-R and a probe Cje-P for specifically detecting the hipO gene of Campylobacter jejuni, wherein the primer sequences are shown in SEQ ID NO.25-26, and the probe sequence is shown in SEQ ID NO.27;
[0018] A forward primer Cbo-F, a reverse primer Cbo-R and a probe Cbo-P for specifically detecting the NTNH gene of Clostridium botulinum, wherein the primer sequences are shown in SEQ ID NO.28-29, and the probe sequence is shown in SEQ ID NO.30;
[0019] A forward primer Pro-F, a reverse primer Pro-R and a probe Pro-P for specifically detecting the atpD gene of Proteus, wherein the primer sequences are shown in SEQ ID NO.31-32, and the probe sequence is shown in SEQ ID NO.33;
[0020] A forward primer RVA-F, a reverse primer RVA-R and a probe RVA-P for specifically detecting the NSP3 gene of group A rotavirus, wherein the primer sequences are shown in SEQ ID NO.34-35, and the probe sequence is shown in SEQ ID NO.36;
[0021] A forward primer GⅠ-F, a reverse primer GⅠ-R and a probe GⅠ-P for specifically detecting the Norovirus GⅠ type ORF gene, wherein the primer sequences are shown in SEQ ID NO.37-38, and the probe sequence is shown in SEQ ID NO.39;
[0022] A forward primer GⅡ-F, a reverse primer GⅡ-R and a probe GⅡ-P for specifically detecting the Norovirus GⅡ type ORF gene, wherein the primer sequences are shown in SEQ ID NO.40-41, and the probe sequence is shown in SEQ ID NO.42;
[0023] A forward primer Ast-F, a reverse primer Ast-R and a probe Ast-P for specifically detecting the astrovirus ORF2 gene, wherein the primer sequences are shown in SEQ ID NO.43-44, and the probe sequence is shown in SEQ ID NO.45;
[0024] A forward primer SV-F, a reverse primer SV-R and a probe SV-P for specifically detecting the Rdrp gene of the safarivirus, wherein the primer sequences are shown in SEQ ID NO.46-47, and the probe sequence is shown in SEQ ID NO.48;
[0025] The forward primer Ead-F, the reverse primer Ead-R and the probe Ead-P for specifically detecting the hexon gene of enteric adenovirus, the primer sequences are shown in SEQ ID NO.49-50, and the probe sequence is shown in SEQ ID NO.51.
[0026] In some embodiments of the present invention, the 5' end of the probe is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group. The fluorescent reporter group includes FAM, VIC, ROX and CY5, and the fluorescent quencher group includes BHQ1, BHQ2 and BHQ3.
[0027] In some embodiments of the present invention, the primers and probes can achieve better detection sensitivity at specific concentrations and combinations, and the concentrations are shown in Table 1. The nucleic acid detection composition with nucleotide sequences as shown in SEQ ID NOs. 1 to 12 in the primer-probe mixture is set as the first group, the nucleic acid detection composition with nucleotide sequences as shown in SEQ ID NOs. 13 to 24 is set as the second group, the nucleic acid detection composition with nucleotide sequences as shown in SEQ ID NOs. 25 to 33 is set as the third group, the nucleic acid detection composition with nucleotide sequences as shown in SEQ ID NOs. 34 to 42 is set as the fourth group, and the nucleic acid detection composition with nucleotide sequences as shown in SEQ ID NOs. 43 to 51 is set as the fifth group.
[0028] A kit comprises the primer-probe combination.
[0029] In some embodiments of the present invention, the kit also includes an enhanced PCR buffer, and the components of the enhanced PCR buffer are as follows: reverse transcriptase, at a concentration of 10U / μL; UDG enzyme, at a concentration of 0.02U / μL; hot start Taq enzyme, at a concentration of 0.5U / μL; Tris-HCl, pH 8.3, at a concentration of 60mM; potassium chloride, at a concentration of 30mM; magnesium chloride, at a concentration of 4mM; betaine, at a concentration of 0.5mM; BSA, at a concentration of 80μg / mL; glycerol, at a concentration of 6%.
[0030] By adopting the above technical solution, multi-target detection is achieved through quadruple fluorescent quantitative PCR, which achieves the purpose of efficiently detecting pathogenic microorganisms in 17 foods, solving the problem that existing in vitro diagnostic kits cannot detect these 17 pathogenic microorganisms at the same time.
[0031] In some embodiments of the present invention, a method for simultaneously detecting 17 pathogenic microorganisms in food, using the kit described in the above scheme and using fluorescent quantitative PCR method, comprises the following steps:
[0032] S1. Extract nucleic acid from samples;
[0033] S2. using a kit to perform an amplification reaction on the nucleic acid of the sample;
[0034] S3. Determine the positive or negative nature of the test sample based on the fluorescence signal pathway and fluorescence intensity.
[0035] In some embodiments of the present invention, the final concentration of the amplification reaction system is composed of: enhanced PCR buffer, 0.1-1 μM forward primer, 0.1-1 μM reverse primer, 0.05-0.5 μM probe and more than 1.0 ng / μL template nucleic acid, and RNase-Free ddH2O is added to 20 μL.
[0036] In some embodiments of the present invention, on the one hand, the kit uses one-step RT-qPCR for detection on a fluorescent quantitative PCR instrument, and the reaction conditions are: Stage 1: 37°C, 2min; Stage 2: 95°C, 30s; Stage 3: (95°C, 10s, 60°C, 30s), 45 cycles of 60°C, collecting fluorescence signals at 30s, and automatically reading once after each cycle. On the other hand, without affecting the detection performance, Stage 1 and Stage 2 can adjust the heating and cooling rate of the PCR instrument to 5°C / s, and Stage 3 can adjust the heating and cooling rate of the PCR instrument to 3°C / s. In order to achieve a faster detection speed and greatly reduce the detection time.
[0037] Preferably, the presence of Salmonella, Staphylococcus aureus, Clostridium perfringens, Escherichia coli O157, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, Vibrio cholerae, Campylobacter jejuni, Clostridium botulinum, Proteus, Group A rotavirus, Norovirus GⅠ, Norovirus GⅡ, Astrovirus, Saruvirus and Enteric adenovirus is determined based on the detected amplification curve and Ct value. The interpretation method is: if there is an S-type amplification curve and the Ct value is less than 38, the sample is positive; if there is no amplification curve, or the Ct value is greater than 38, the sample is negative.
[0038] The detection range for Salmonella, Escherichia coli O157, Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio cholerae, and Campylobacter jejuni is 500-1000CFU / mL; the detection range for Staphylococcus aureus, Clostridium botulinum, and Proteus is 1000-2000CFU / mL; the detection range for Clostridium perfringens and Bacillus cereus is 2500-5000CFU / mL; the detection range for group A rotavirus, norovirus GⅠ, norovirus GⅡ, astrovirus, saprovirus, and enteric adenovirus is 250-500copies / mL.
[0039] By adopting the above technical scheme, the method of the present invention is simple and rapid to operate, and the result is highly accurate, eliminating the step of electrophoresis after the traditional PCR reaction. This provides a convenient, rapid and effective technical means for in vitro diagnosis of pathogenic microorganisms in food, and helps the kit mentioned in the above scheme to perform large-scale screening in the field of food safety.
[0040] Compared with the prior art, the invention has the following beneficial effects: the detection method is simple and time-saving; the detection results have the advantages of high sensitivity, strong specificity and good repeatability, and can be used for large-scale screening of pathogenic microorganisms in food. The qPCR method has the advantages of simple operation and equipment, and the experiment is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 This is a comparison of the primers and probes for Bacillus cereus in Example 1 before and after modification.
[0042] Figure 2 This is the amplification curve diagram of the first group in Example 1.
[0043] Figure 3 This is the amplification curve of the second group in Example 1.
[0044] Figure 4 This is the amplification curve diagram of the third group in Example 1.
[0045] Figure 5 This is the amplification curve diagram of the fourth group in Example 1.
[0046] Figure 6 This is the amplification curve of the fifth group in Example 1. DETAILED DESCRIPTION
[0047] In a single tube reaction, multiple targets are specifically amplified at the same time. The entire qRT-PCR system is much more complicated than the single target amplification system. There are many factors that affect the effect of multiple qRT-PCR reactions, which can be roughly divided into two categories: qRT-PCR system and reaction conditions. Among them, the qRT-PCR system mainly includes primer and probe combinations, enzyme mixture and PCR buffer, and the reaction conditions mainly include annealing temperature and number of cycles.
[0048] The present invention will be further described in detail with reference to the examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The materials, reagents, instruments and equipment used in the following examples are all commercially available unless otherwise specified. The reagents used are all analytically pure and the water is nuclease-free pure water.
[0049] Example 1
[0050] 1. Design and combination of specific primers and probes for 17 food pathogenic microorganisms
[0051] The design of primers and probes is the key to multiplex qRT-PCR, which directly affects the sensitivity and specificity of multiplex detection. If the primers and probes are not designed properly, they are likely to form dimers or non-specific amplification, which not only consumes the components in the reaction system but also affects the annealing and extension rates. Generally, the more targets there are, the more difficult it is to design primers and probes.
[0052] When designing primers and probes, first determine the appropriate conservative target gene by searching the literature, then download the target gene sequence from Genebank, perform conservative analysis and review through Blast comparison, screen highly conservative regions as the preferred targets for primer and probe design, and then use Oligo7 to design specific primers and probes, and perform sequence comparison at the National Center for Biotechnology Information (NCBI) to prove the specificity of each primer. It is necessary to consider the reaction kinetics of primers and probes, similar Tm values, similar GC content, and the inability to form dimers between primers. Finally, a database homology comparison should be performed to ensure the specificity of each primer amplification. Some target genes have high homology. In order to ensure the conservatism of primers, it is necessary to re-screen the conservative sequence design or modify the designed primers and probes to improve the specificity of detection. After the primers are designed, they are sent to the company for synthesis. After the primers are synthesized, qRT-PCR needs to be used to verify the specificity of the primers and probes, the amplification efficiency, and the amplification curve line shape.
[0053] The following shows examples of primer and probe modifications during the design process:
[0054] When designing primers and probes for Bacillus cereus, a forward primer of 5'-CCAGAAACTTTATAACCGCC-3', a reverse primer of 5'-AYGTTAGTATCGAAGAAGATAATAGTATTC-3', and a probe of 5'-CCATTTTTTCTTGTATACCAACYGGAATACC-3' were selected. However, when the sequences were specifically aligned, it was found that there were overlaps with Bacillus anthracis, Bacillus thuringiensis, and Bacillus albicans. In the actual test process, it was also found that when detecting Bacillus thuringiensis, an amplification curve (such as Figure 1 As all of them belong to the genus Bacillus, there is no suitable target gene to distinguish them. Therefore, the locked nucleic acid technology was used to modify the locked nucleic acid using the specific sites that distinguish Bacillus cereus from other Bacillus. After the modification, there was no amplification curve for detecting Bacillus thuringiensis (as shown in Figure 1 Therefore, the detection of Bacillus cereus has a high specificity.
[0055] As the number of detection targets in the composite amplification system increases, it becomes more difficult to control the amplification balance of each target due to competition among the amplification primers of each target. If any combination test shows non-specificity or decreased amplification efficiency or poor amplification curve, it is necessary to adjust the detection targets, or adjust the concentration and ratio of each target primer and probe, or even redesign the primers and probes until all combinations meet the requirements of specificity, efficiency and peak type of amplification products.
[0056] Through a large number of repeated single-tube multiplex combination tests, the detection sequences (Table 1) and detection combinations (Table 2) of primers and probes for 17 food pathogenic microorganisms were finally determined. The amplification curves of each detection combination are shown in Figure 2-6 shown.
[0057] Table 1 Primer and probe sequences and final concentrations of 17 food pathogenic microorganisms
[0058]
[0059]
[0060]
[0061] Table 2 Detection combination of 17 food pathogenic microorganisms
[0062]
[0063] 2. Enhanced PCR buffer optimization
[0064] The reverse transcriptase, DNA polymerase, UDG enzyme, buffer, MgCl2, dNTP concentration and additives in the PCR reaction system will affect the test results. Reverse transcriptase is used to reverse transcribe viral RNA nucleic acid into cDNA; DNA polymerase is used to catalyze PCR reaction. Since there are multiple target amplification reactions in multiplex PCR, each target amplification will compete with DNA polymerase to varying degrees. If the final concentration of DNA polymerase is too low, it will inhibit the amplification of low-efficiency targets, and too much will lead to nonspecific amplification. In order to enhance the specificity and sensitivity of PCR amplification, appropriate MgCl2, dNTP concentrations and additives need to be added to the PCR buffer. The ions and additive components in the PCR buffer can only play their greatest role at their optimal reaction concentration. If the concentration is too low, the effect is not significant. On the contrary, it will inhibit PCR amplification. Therefore, it is necessary not only to select appropriate PCR components, but also to optimize the concentrations of various components of the PCR reaction system. In addition, the UDG system is also introduced into the PCR reaction system to prevent aerosol contamination of RCR products in the environment. The final enhanced PCR buffer optimization results are shown in Table 3.
[0065] Table 3 PCR buffer components and their final concentrations
[0066] Components concentration Reverse transcriptase 10U / μL HS Taq 0.5U / μL UDG enzyme 0.02U / μL Tris-HCl, pH 8.3 60mM Potassium chloride 30mM Magnesium chloride 4mM Betaine 0.5mM BSA 80 μg / mL glycerin 6％
[0067] 3. Optimization of PCR amplification program
[0068] Generally, PCR amplification needs to go through a cycle of three steps, namely, denaturation, annealing, and extension, in order to enrich the target fragment. The present invention uses a rapid PCR amplification program, combines the annealing and extension steps into one, changes the three-step program into a two-step program, and reduces the reaction time of each step. Annealing temperature is an important factor affecting the specificity of PCR reaction. Within a certain temperature range, the higher the annealing temperature, the higher the amplification specificity. The lower the annealing temperature, the lower the amplification specificity. Although lowering the annealing temperature may increase the amplification yield, the mismatch between the primer and the template will also increase, resulting in an increase in nonspecific amplification. On the contrary, although increasing the annealing temperature can improve the specificity of the reaction, it will cause a decrease in amplification efficiency, or even the appearance of no amplified product. The present invention optimizes the amplification time and annealing temperature, and the specific amplification program is shown in Table 4 below.
[0069] In addition, in order to improve the detection efficiency and shorten the overall detection time, the present invention adjusts and optimizes the heating and cooling rates of the instrument in the reaction program while ensuring the detection performance. The optimized amplification program is shown in Table 5.
[0070] Table 4 Optimized PCR amplification program
[0071]
[0072] Note: 1 Fluorescence signals were collected at the end of this period.
[0073] Table 5 PCR amplification rapid procedure 1
[0074]
[0075] Note: 1 Suitable for fluorescent quantitative PCR instruments with adjustable heating and cooling rates, such as SLAN96, Gentier96, etc.
[0076] 2 Fluorescence signals were collected at the end of this period.
[0077] 4. Results Analysis
[0078] The presence of Salmonella, Staphylococcus aureus, Clostridium perfringens, Escherichia coli O157, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, and Vibrio cholerae is determined based on the detected amplification curve and Ct value. The interpretation method is: if there is an S-type amplification curve and the Ct value is less than 38, the sample is positive; if there is no amplification curve, or the Ct value is greater than 38, the sample is negative.
[0079] Example 2
[0080] Sample testing and specificity studies
[0081] (1) Sample selection
[0082] The sample to be tested in this embodiment is a commercial standard strain, and of course, it can also be a culture that has been clinically identified and isolated.
[0083] (2) Nucleic acid extraction
[0084] The samples were extracted using commercial magnetic beads or silica gel mold methods.
[0085] (3) qPCR amplification reaction
[0086] The reaction system was prepared according to the final concentration of each component in Table 1 and Table 2, wherein SEQ ID NO.1-12 was the first group detection system, SEQ ID NO.13-24 was the second group detection system, SEQ ID NO.25-33 was the third group detection system, SEQ ID NO.34-42 was the fourth group detection system, and SEQ ID NO.43-51 was the fifth group detection system. 5 μL of template DNA was added, and RNase-Free ddH2O was used to make up to 20 μL. The reaction solution was mixed evenly and transferred to a PCR reaction tube, and the tube cap was covered.
[0087] The prepared system was run on a fluorescent quantitative PCR instrument with the following reaction conditions: Stage 1: 37°C, 2 min; Stage 2: 95°C, 30 s; Stage 3: (95°C, 10 s, 60°C, 30 s), 45 cycles, collecting fluorescence signals at 60°C, 30 s, and automatically reading once after each cycle.
[0088] (4) Result determination
[0089] The results are automatically saved at the end of the reaction. Adjust the Start value, End value, and Threshold value of the Baseline according to the analyzed image (the Start value can be set between 3-15, and the End value can be set between 5-20, and the amplification curve of the negative control can be adjusted to be flat or below the threshold line). Click Analysis to automatically obtain the analysis results and view the results in the Report interface.
[0090] The detailed judgment method and standards are as follows: (1) When the positive control shows an "S"-shaped amplification curve and the Ct value is ≤38, and the negative control has no S-shaped amplification curve or the Ct value is >38, it indicates that there is no problem with the entire detection process and the detection result of the sample to be tested is credible; (2) When the condition (1) is met, the sample to be tested shows an "S"-shaped amplification curve and the Ct value is ≤38, indicating that the sample to be tested is positive; (3) When the condition (1) is met, the sample to be tested does not show an "S"-shaped amplification curve or the Ct value is >38, indicating that the sample to be tested is negative.
[0091] On the contrary, when the positive control lacks a Ct value or an "S"-shaped amplification curve, or the negative control has a Ct value and an "S"-shaped amplification curve, it indicates that there is a problem with the entire detection process, or there is contamination, and the test results of the sample to be tested are unreliable, and the test operation needs to be repeated to further determine the situation in the sample to be tested.
[0092] Table 6 Detection results of the commercial standard strains using the present invention
[0093] sample serial number Ct value Interpretation of results Shigella ATCC 12038 No Ct Negative Vibrio alginolyticus BNCC337013 No Ct Negative Vibrio fluvialis CGMCC 1.1608 No Ct Negative Aeromonas hydrophila CGMCC 1.1801 No Ct Negative Pleistomonas shigelloides CICC 10380 No Ct Negative Cronobacter sakazakii CICC 21645 No Ct Negative Clostridium difficile ATCC 43255 No Ct Negative Enterococcus faecalis ATCC 51299 No Ct Negative Bacillus mycoides ATCC 10206 No Ct Negative Bacillus suivrei ATCC 10792 No Ct Negative Bacillus megaterium ATCC 14581 No Ct Negative
[0094] (5) Results analysis
[0095] The test results showed that the standard strains of similar species purchased by the kit were all negative, indicating that there was no cross reaction. Therefore, the kit has excellent analytical specificity.
[0096] Example 3
[0097] Sensitivity studies
[0098] The samples to be tested in this embodiment are commercial standard strains or pseudoviruses. The strains are gradient diluted to 1×10 5 cfu / ml, 1×10 4 cfu / ml, 5×10 3 cfu / ml, 2000 cfu / ml, 1000 cfu / ml, 500 cfu / ml, 250 cfu / ml, and the pseudovirus was diluted to 1×10 4 The detection sensitivity of 17 kinds of food pathogenic microorganisms is as follows: the detection range of Salmonella (ATCC 9120), Escherichia coli O157 (CMCC (B) 45939), Listeria monocytogenes (ATCC 19115), Vibrio parahaemolyticus (CMCC (B) 20033), Vibrio cholerae (CMCC (B) 17247), Campylobacter jejuni (CICC 22936) is 500-1000CFU / mL; the detection range of Staphylococcus aureus (ATCC 9144), Clostridium botulinum (GDMCC 1.3725), Proteus (ACCC The detection range of the test kit for 11002) is 1000-2000 CFU / mL; the detection range of Clostridium perfringens (CMCC(B)64724) and Bacillus cereus (ATCC 2) is 2500-5000 CFU / mL; the detection range of group A rotavirus, norovirus GⅠ, norovirus GⅡ, astrovirus, saprovirus, and enteroadenovirus is 250-500 copies / mL. The details are shown in Table 7.
[0099] Table 7 Sensitivity test results of 17 food pathogenic microorganisms
[0100]
[0101]
Claims
1. A set of primer-probe combinations for simultaneously detecting pathogenic microorganisms in 17 foods, characterized in that: Includes primer and probe combinations for detecting Salmonella, Staphylococcus aureus, Clostridium perfringens, Escherichia coli O157, Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus, Vibrio cholerae, Campylobacter jejuni, Clostridium botulinum, Proteus, group A rotavirus, norovirus GⅠ, norovirus GⅡ, astrovirus, saprovirus, and enteric adenovirus; in: A forward primer Sal-F, a reverse primer Sal-R and a probe Sal-P for specifically detecting the Salmonella invA gene, wherein the primer sequences are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.3; A forward primer Cpe-F, a reverse primer Cpe-R and a probe Cpe-P for specifically detecting the plc gene of Clostridium perfringens, wherein the primer sequences are shown in SEQ ID NO.4-5, and the probe sequence is shown in SEQ ID NO.6; A forward primer Esc-F, a reverse primer Esc-R and a probe Esc-P for specifically detecting the rfbE gene of Escherichia coli O157, wherein the primer sequences are shown in SEQ ID NO.7-8, and the probe sequence is shown in SEQ ID NO.9; A forward primer Vpa-F, a reverse primer Vpa-R and a probe Vpa-P for specifically detecting the toxS gene of Vibrio parahaemolyticus, wherein the primer sequences are shown in SEQ ID NO.10-11, and the probe sequence is shown in SEQ ID NO.12; A forward primer Sau-F, a reverse primer Sau-R and a probe Sau-P for specifically detecting the nuc gene of Staphylococcus aureus, wherein the primer sequences are shown in SEQ ID NO.13-14, and the probe sequence is shown in SEQ ID NO.15; A forward primer Vch-F, a reverse primer Vch-R and a probe Vch-P for specifically detecting the hlyA gene of Vibrio cholerae, wherein the primer sequences are shown in SEQ ID NO.16-17, and the probe sequence is shown in SEQ ID NO.18; A forward primer Bac-F, a reverse primer Bac-R and a probe Bac-P for specifically detecting the cerB gene of Bacillus cereus, wherein the primer sequences are shown in SEQ ID NO.19-20, and the probe sequence is shown in SEQ ID NO.21; A forward primer Lmo-F, a reverse primer Lmo-R and a probe Lmo-P for specifically detecting the actA gene of Listeria monocytogenes, wherein the primer sequences are shown in SEQ ID NO.22-23, and the probe sequence is shown in SEQ ID NO.24; A forward primer Cje-F, a reverse primer Cje-R and a probe Cje-P for specifically detecting the hipO gene of Campylobacter jejuni, wherein the primer sequences are shown in SEQ ID NO.25-26, and the probe sequence is shown in SEQ ID NO.27; A forward primer Cbo-F, a reverse primer Cbo-R and a probe Cbo-P for specifically detecting the NTNH gene of Clostridium botulinum, wherein the primer sequences are shown in SEQ ID NO.28-29, and the probe sequence is shown in SEQ ID NO.30; A forward primer Pro-F, a reverse primer Pro-R and a probe Pro-P for specifically detecting the atpD gene of Proteus, wherein the primer sequences are shown in SEQ ID NO.31-32, and the probe sequence is shown in SEQ ID NO.33; A forward primer RVA-F, a reverse primer RVA-R and a probe RVA-P for specifically detecting the NSP3 gene of group A rotavirus, wherein the primer sequences are shown in SEQ ID NO.34-35, and the probe sequence is shown in SEQ ID NO.36; A forward primer GⅠ-F, a reverse primer GⅠ-R and a probe GⅠ-P for specifically detecting the Norovirus GⅠ type ORF gene, wherein the primer sequences are shown in SEQ ID NO.37-38, and the probe sequence is shown in SEQ ID NO.39; A forward primer GⅡ-F, a reverse primer GⅡ-R and a probe GⅡ-P for specifically detecting the Norovirus GⅡ type ORF gene, wherein the primer sequences are shown in SEQ ID NO.40-41, and the probe sequence is shown in SEQ ID NO.42; A forward primer Ast-F, a reverse primer Ast-R and a probe Ast-P for specifically detecting the astrovirus ORF2 gene, wherein the primer sequences are shown in SEQ ID NO.43-44, and the probe sequence is shown in SEQ ID NO.45; A forward primer SV-F, a reverse primer SV-R and a probe SV-P for specifically detecting the Rdrp gene of the safarivirus, wherein the primer sequences are shown in SEQ ID NO.46-47, and the probe sequence is shown in SEQ ID NO.48; The forward primer Ead-F, the reverse primer Ead-R and the probe Ead-P for specifically detecting the hexon gene of enteric adenovirus, the primer sequences are shown in SEQ ID NO.49-50, and the probe sequence is shown in SEQ ID NO.
51.
2. The primer-probe combination according to claim 1, characterized in that The 5' end of the probe is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group.
3. The primer-probe composition according to claim 2, characterized in that: The fluorescent reporter groups include FAM, VIC, ROX and CY5, and the fluorescent quencher groups include BHQ1, BHQ2 and BHQ3.
4. The primer-probe combination according to claim 1, characterized in that: In the primer-probe composition, the primers and probe combinations with nucleotide sequences as shown in SEQ ID NOs. 1 to 12 are set as the first group, the primers and probe combinations with nucleotide sequences as shown in SEQ ID NOs. 13 to 24 are set as the second group, the primers and probe combinations with nucleotide sequences as shown in SEQ ID NOs. 25 to 33 are set as the third group, the primers and probe combinations with nucleotide sequences as shown in SEQ ID NOs. 34 to 42 are set as the fourth group, and the primers and probe combinations with nucleotide sequences as shown in SEQ ID NOs. 43 to 51 are set as the fifth group.
5. A kit, characterized in that: The invention comprises the primer-probe combination as claimed in claim 1.
6. The kit according to claim 5, characterized in that The kit also includes an enhanced PCR buffer, wherein the enhanced PCR buffer reaction components include: reverse transcriptase at a concentration of 10 U / μL; UDG enzyme at a concentration of 0.02 U / μL; hot start Taq enzyme at a concentration of 0.5 U / μL; Tris-HCl at pH 8.3 at a concentration of 60 mM; potassium chloride at a concentration of 30 mM; magnesium chloride at a concentration of 4 mM; betaine at a concentration of 0.5 mM; BSA at a concentration of 80 μg / mL; and glycerol at a concentration of 6%.
7. A method for simultaneously detecting pathogenic microorganisms in 17 kinds of food, characterized in that: The following steps are involved: S1. Extract nucleic acid from samples; S2. using the primer-probe combination of claim 1 or the kit of claim 5 to amplify the nucleic acid of the sample; S3. Determine the positive or negative nature of the test sample based on the fluorescence signal pathway and fluorescence intensity.
8. The method according to claim 7, characterized in that The amplification reaction system consists of: enhanced PCR buffer, 0.1-1 μM forward primer, 0.1-1 μM reverse primer, 0.05-0.5 μM probe and more than 1.0 ng / μL template nucleic acid, and RNase-Free ddH2O is added to 20 μL.
9. The method according to claim 7, characterized in that: The conditions of the amplification reaction are as follows: Stage 1: 37°C, 2 min; Stage 2: 95°C, 30 s; Stage 3: 95°C, 10 s, 60°C, 30 s, 45 cycles, collecting fluorescence signals at 60°C, 30 s, and automatically reading once after each cycle.
10. The method according to claim 7, characterized in that The method of judging the positive or negative nature of the test sample based on the fluorescence signal pathway and fluorescence intensity is specifically: if there is an S-type amplification curve and the Ct value is less than 38, the sample is positive; if there is no amplification curve, or the Ct value is greater than 38, the sample is negative.