Primer combination for multiple detection kit of food-borne pathogenic bacteria and multiple detection kit

Abstract

The invention provides a primer combination of a multiple detection kit for food-borne pathogenic bacteria and the multiple detection kit. The primer combination comprises probes and primers for detecting nine food-borne pathogenic bacteria: vibrio cholerae general type, vibrio cholerae O1 type, vibrio cholerae O139 type, vibrio parahaemolyticus, salmonella, yersinia enterocolitica, campylobacter, listeria monocytogenes and enterobacter sakakaakii, and the sequence is shown as SEQ1-31; the probe and the primer pair are used for detecting nine characteristic genes, namely a shigella universal type: ipaH, a diarrheogenic Escherichia coli universal type: eltA, sta3, sta and aggR, and enterohemorrhagic Escherichia coli: stx1, stx2, escV and rfbE, and the sequences of the probe and the primer pair are shown as SEQ32-64. According to the invention, by combining a digital PCR multiple coding technology, the detection of 12 food-borne pathogenic bacteria can be realized, and the sensitivity, the specificity and the accuracy are relatively good.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly to a primer combination and a multiplex detection kit for a multiplex detection kit of foodborne pathogenic bacteria. Background Art

[0002] Foodborne pathogens are the main factors causing foodborne diseases, and refer to a class of pathogenic bacteria that cause food contamination and human diseases during the processes of food processing and circulation. The most common pathogenic bacteria include Listeria monocytogenes (abbreviated as L. monocytogenes), Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica, Bacillus cereus, Vibrio spp., Campylobacter jejuni, and Shiga toxin-producing Escherichia coli (STEC), etc.

[0003] Outbreaks and sporadic cases of foodborne diseases often occur worldwide. Due to their high incidence and mortality rates, they cause serious economic burdens and have become a major global public health problem. Therefore, the qualitative and quantitative detection of foodborne pathogenic bacteria can ensure food safety and reduce the occurrence of foodborne diseases. Thus, a rapid, sensitive, and efficient detection technology is crucial for ensuring food safety.

[0004] Existing detection methods for foodborne pathogenic bacteria include traditional culture and biochemical identification methods, digital PCR, multiplex PCR / qPCR, biosensors, etc., but there are still problems such as long time consumption, low sensitivity, high cost, and poor stability. Therefore, further research on the multiplex detection of foodborne pathogenic bacteria is still needed. Summary of the Invention

[0005] Aiming at the problems of poor stability, long time consumption, high cost, and low sensitivity existing in the prior art, the purpose of the present invention is to provide a primer combination and a multiplex detection kit for a multiplex detection kit of foodborne pathogenic bacteria.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The present invention provides a primer combination for multiplex detection of foodborne pathogenic bacteria, and the primer combination includes: primer combination A and primer combination B;

[0008] The primer combination A includes: probes and primer pairs for detecting 9 foodborne pathogenic bacteria; the 9 foodborne pathogenic bacteria include: Vibrio cholerae general type, Vibrio cholerae O1 type, Vibrio cholerae O139 type, Vibrio parahaemolyticus, Salmonella, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Enterobacter sakazakii; the probes and primer pairs in the primer combination A are shown as sequences: SEQ1-SEQ31;

[0009] The primer combination B includes: probes and primer pairs for detecting 9 characteristic genes; the 9 characteristic genes include: Shigella genus general type: ipaH, diarrheagenic Escherichia coli general type: eltA, sta3, sta, aggR, enterohemorrhagic Escherichia coli: stx1, stx2, escV, rfbE; the probes and primer pairs in the primer combination B are shown as sequences: SEQ32-SEQ64.

[0010] Preferably, the probe is labeled with a fluorescent group or a quenching group at the 5'-end and / or 3'-end.

[0011] Preferably, the fluorescent group is any one or more of FAM, HEX, ROX, CY5, Q705; the quenching group is any one or more of BHQ1, BHQ2, BHQ3.

[0012] Preferably, in order to achieve multiplex detection, the probes for detecting the same foodborne pathogenic bacterium or characteristic gene include probes labeled with different fluorescent groups.

[0013] Preferably, the fluorescence labeling of the probes in the primer combination A is shown in Table 1, and the fluorescence labeling of the probes in the primer combination B is shown in Table 2.

[0014] The present invention provides a kit for multiplex detection of foodborne pathogenic bacteria, and the kit includes the primer combination described above.

[0015] Preferably, the kit further includes a detection Buffer, a detection enzyme, a positive control product, and a negative control product.

[0016] Preferably, the detection Buffer is dPCR Buffer; the detection enzyme is dPCRTaq enzyme; the positive control product is a mixed solution of artificial synthetic genes containing the target sequences of the foodborne pathogenic bacteria; the negative control product is nuclease-free water.

[0017] The present invention provides a detection method for multiplex detection of foodborne pathogenic bacteria, and the detection method includes the following steps:

[0018] (1) Extract the DNA of the sample to be tested;

[0019] (2) Perform digital PCR reactions using the primer combinations A and B respectively;

[0020] (3) Combine the multi-color fluorescence encoding and decoding technology to perform genotyping detection on the results.

[0021] Preferably, the primer combination includes primer combination A and primer combination B; when performing digital PCR reaction using primer combination A, the detection channels are selected as FAM, HEX, ROX, and Cy5; when performing digital PCR reaction using primer combination B, the detection channels are selected as FAM, HEX, ROX, Cy5, and Q705.

[0022] Preferably, in the digital PCR reaction system, the final concentration of each probe in the primer combination is 100 - 400 nM, and the final concentration of each primer is 400 - 600 nM.

[0023] Preferably, the sample to be tested is a food sample or a human fecal sample.

[0024] Advantages of the technical solution of the present invention:

[0025] The present invention provides a primer combination and a multiplex detection kit for foodborne pathogenic bacteria multiplex detection. Combining the digital PCR multiplex encoding technology, it can break through the channel limitation and simultaneously detect 12 kinds of foodborne pathogenic bacteria. The primer combination and kit of the present invention have good sensitivity, specificity, and accuracy. Among them, the detection limit for pure cultures of 12 kinds of foodborne pathogenic bacteria is 0.6 copies / μL (qPCR is 100 copies / μL), with excellent sensitivity; through BLAST comparison and experimental verification, the present invention has no cross-reaction with more than 50 non-target bacteria (such as lactic acid bacteria, probiotics), with good specificity; in the clinical test of 12 samples, the consistency between the method of the present invention and the culture method results reaches 100%. Description of the Drawings

[0026] Figure 1 It is an example of the four-color fluorescence encoding detection results of FAM, HEX, ROX, and CY5 of the present invention;

[0027] In the figure, all positive points in the four channels are located at the same position, that is, it is determined that the template to be tested is the template corresponding to the 4-color encoded target;

[0028] Figure 2 It is an example of the results of the FAM single-color encoded target and the HEX + ROX double-color encoded target of the present invention;

[0029] In the figure, the positive points in the HEX and ROX channels are located at the same position, and it can be confirmed as the detection result of the double-color encoded target. The positive points in the FAM channel are at different positions from the positive points in the HEX and ROX channels. Therefore, it is confirmed as the detection result of the FAM channel single-color encoded target;

[0030] Figure 3 Schematic diagram of the 2-channel one-dimensional scatter plot in Embodiment 3 of the present invention;

[0031] Figure 4 Schematic diagram of Type I of the FAM and HEX two-dimensional scatter plot in Embodiment 3 of the present invention;

[0032] Figure 5 Schematic diagram of Type II of the FAM and HEX two-dimensional scatter plot in Embodiment 3 of the present invention.

[0033] Figure 6A Test result of the negative sample of Primer Combination A of the present invention.

[0034] Figure 6B Test result of the negative sample of Primer Combination B of the present invention.

[0035] Figure 7 Positive one-dimensional scatter plot of the linearity test in Experimental Example 1.

[0036] Figure 8 Linearity curve of the linearity test in Experimental Example 1.

[0037] Figure 9 Research result of the primer-probe optimization designed for Vibrio parahaemolyticus.

[0038] Figure 10 Research result of the primer-probe optimization designed for the general type of Vibrio cholerae.

[0039] Figure 11 Research result of the primer-probe optimization designed for Listeria monocytogenes.

[0040] Figure 12 Research result of the primer-probe optimization designed for the general type characteristic gene ipaH of Shigella.

[0041] Figure 13 Research result of the primer-probe optimization designed for the general type characteristic gene stx1 of diarrheagenic Escherichia coli.

[0042] Figure 14 Research result of the primer-probe optimization designed for the characteristic gene rfbE of enterohemorrhagic Escherichia coli (EHEC) (O157). Specific Embodiments

[0043] The following further elaborates on the specific embodiments of the present invention in conjunction with the embodiments. Unless otherwise specified, the instruments and equipment involved in the embodiments are all conventional instruments and equipment; the raw materials involved are all commercially available conventional raw materials; the test methods involved are all conventional methods.

[0044] Embodiment 1 Design of Primer-Probe System

[0045] The primer combination and multiplex detection kit of the present invention for the multiplex detection kit of foodborne pathogenic bacteria can achieve the quantitative detection of 12 foodborne pathogenic bacteria, including Vibrio cholerae general type, Vibrio cholerae O1 type, Vibrio cholerae O139 type, Vibrio parahaemolyticus, Salmonella, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Cronobacter sakazakii, Shigella general type, diarrheagenic Escherichia coli general type, enterohemorrhagic Escherichia coli (EHEC) (O157). Among them, the diarrheagenic Escherichia coli general type includes 5 pathogenic bacteria: enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), and enteroaggregative Escherichia coli (EAEC). By detecting any one or more characteristic genes among the above 5 pathogenic bacteria, the detection of the diarrheagenic Escherichia coli general type can be achieved. For the detection of enterohemorrhagic Escherichia coli (EHEC) (O157), in addition to detecting the characteristic genes, it is also necessary to detect the O antigen characteristic genes of the O157 serotype.

[0046] (1) Primer combination A is used for the identification of the species and genus of specific foodborne pathogenic bacteria, including the detection of Vibrio cholerae general type (or simply referred to as Vibrio cholerae), Vibrio cholerae O1 type, Vibrio cholerae O139 type, Vibrio parahaemolyticus, Salmonella, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, and Cronobacter sakazakii, a total of 9 pathogenic bacteria. The primer combination is designed according to the conserved regions of foodborne pathogenic bacteria (as shown in Table 1), and the specific method is as follows: ① Download the whole genome dataset of the species from NCBI; ② Screen for species-specific genes; ③ Analyze the conservation of specific genes; ④ Design primers and probes; ⑤ Analyze the specificity and dimer of multiplex primers and probes according to MFE Primer.

[0047] (2) Primer combination B is used for the detection of Shigella general type, diarrheagenic Escherichia coli general type, and enterohemorrhagic Escherichia coli (EHEC) (O157). Among them, referring to the document "GB 4789.6-2016 National Food Safety Standard Microbiological Examination of Foods - Examination of Diarrheagenic Escherichia coli", primer combination B is designed according to the characteristic genes given in the document, and the specific primer and probe information is shown in Table 2.

[0048] Table 1 Sequence information corresponding to primer combination A

[0049]

[0050]

[0051] Table 2 Sequence information corresponding to primer combination B

[0052]

[0053]

[0054] Note: For EHEC / EPEC, the interpretation should be based on the negative / positive combination results of the detected characteristic genes.

[0055] Table 3 presents the coverage rates of primer pair A and primer pair B in the whole-genome datasets of the corresponding targets in the NCBI database. It can be seen that the coverage rate of the primer-probe combinations designed in the present invention for the selected foodborne pathogenic bacteria basically reaches over 98%.

[0056] Table 3 Target Coverage Rates of Primer-Probe Combinations

[0057]

[0058] Example 2 A Kit for Multiplex Detection of Foodborne Pathogenic Bacteria

[0059] The present invention provides a kit for multiplex detection of foodborne pathogenic bacteria, comprising: primer pair A and primer pair B described in Example 1, foodborne pathogenic bacteria detection Buffer (3×Maxuseful dPCR Buffer), foodborne pathogenic bacteria detection enzyme (dPCR Taq enzyme), positive control product (a mixed solution of linearized plasmids containing foodborne pathogenic bacteria target fragments synthesized artificially), negative control product (nuclease-free water), digital PCR chip, digital PCR oil phase A, and digital PCR oil phase B.

[0060] Table 4 Digital PCR Reaction System

[0061] Component Volume Foodborne Pathogen Detection Buffer 10 μL Foodborne Pathogen Detection Enzyme 1 μL Foodborne Pathogen Detection Primer Pair A / Primer Pair B 9 μL Nuclease-Free Water 0 - 5 μL Sample to be Tested / Positive Control / Negative Control 5 - 10 μL Total Volume 30 μL

[0062] Note: The final concentration of each probe in the primer pair is 100 - 200 nM, and the final concentration of each primer is 400 nM.

[0063] Table 5 Digital PCR Reaction Conditions

[0064]

[0065] Example 3 A Detection Method for Multiplex Detection of Foodborne Pathogenic Bacteria

[0066] The present invention provides a detection method for a kit for multiplex detection of foodborne pathogenic bacteria, which combines multi-color fluorescence encoding and decoding technology to perform typing detection on various pathogens or pathogen subtypes. The specific detection method comprises the following steps:

[0067] (1) Extract the DNA of the sample to be detected;

[0068] Extract nucleic acids from 1-2 g of fecal samples. Positive and negative quality control samples are not involved in nucleic acid extraction.

[0069] Extract nucleic acids according to the instruction manual of the Suzhou Beaver Biotech fecal nucleic acid extraction kit (product number: 70411-100). For specific extraction steps, please refer to the corresponding extraction kit instruction manual.

[0070] (2) Use the kit for multiplex detection of foodborne pathogenic bacteria described in Example 2 to prepare digital PCR reaction systems for primer sets A and B respectively.

[0071] Complete the equipment operation according to the instrument operation manual (operation example of the SCI Digital series digital PCR platform). ① Initialization: After turning on the equipment, open the "SCI Digital" software shortcut on the computer desktop. The software will automatically connect to the equipment and complete the equipment initialization; ② Place consumables: Open the equipment chamber door and place the unused digital PCR chip, 12-well strip tube filled with PCR reaction solution, oil phase A, oil phase B, sampling pipette tip, and waste box at the corresponding workstations of the equipment in sequence.

[0072] (3) Combine the multi-color fluorescence encoding and decoding technology to perform genotyping detection on the results.

[0073] Among them, when performing digital PCR reaction with primer set A, select FAM, HEX, ROX, and Cy5 as the detection channels; when performing digital PCR reaction with primer set B, select FAM, HEX, ROX, Cy5, and Q705 as the detection channels.

[0074] (4) Data collection and analysis

[0075] Threshold setting: First, adjust the threshold line in the one-dimensional scatter plot. The threshold line can be automatically set by the software or manually adjusted: just above the highest point of the negative droplets.

[0076] Data analysis: The digital PCR reaction system uses multi-color coding for result interpretation. When there is more than one positive result channel, it is also confirmed whether it is a multiple infection through the two-dimensional scatter plot result. As Figure 3 shown, there are positive signals in FAM and HEX in the one-dimensional scatter plot; the confirmation of multiple infections in the two-dimensional scatter plot is as follows:

[0077] ① When the two-dimensional scatter plot is Figure 4 , the FAM and HEX signals appear in the same chamber, indicating that the positive signal comes from Vibrio cholerae.

[0078] ② When the two-dimensional scatter plot is Figure 5 , the FAM and HEX signals appear in different chambers, indicating that the sample has a multiple infection.

[0079] (5) Positive judgment value

[0080] ① The positive judgment values of each detection target of this kit are shown in Table 6 below:

[0081] Table 6 Positive judgment values of each detection target of this kit

[0082] Serial Number Number of Positive Droplets in FAM / HEX / ROX / Cy5 / Q705 Channels Result Judgment 1 ≥3 Positive 2 ＜3 Negative

[0083] ② According to the positive and negative results of each channel, refer to Table 7-8 below (Note: Only the situation of single pathogen infection is shown in the table) to interpret the detection results of pathogens:

[0084] Table 7

[0085]

[0086] Table 8

[0087]

[0088] Note: "+ / -" means that as long as HEX is positive, whether Q705 is negative or positive indicates that the identification result is Enterohemorrhagic Escherichia coli (EHEC).

[0089] ③ Assign values to the corresponding pathogen detections according to the concentration values of the corresponding channels. The detection limit of 12 foodborne pathogenic bacteria is 600 copies / mL.

[0090] (6) Interpretation of test results

[0091] ① Quality control: The number of positive droplets in the 5 channels of the negative control product is < 3, and the copy number concentration of each channel of the positive control product FAM, HEX, ROX, Cy5, Q705 ≥ 20 copies / μL; otherwise, this experiment is invalid.

[0092] ② Negative result: The number of positive droplets of FAM, HEX, ROX, Cy5, Q705 is < 3.

[0093] ③ Positive result: There are positive droplets in the 5 channels, and the number of droplets ≥ 3. Obtain the copy number concentration in the positive channel in the result list of the data analysis software.

[0094] Experimental Example 1 Test results of negative samples of the primer combination of the present invention

[0095] Using the primer combination designed in Example 1, perform dPCR detection on negative fecal nucleic acid samples or human gDNA samples according to the detection method of Example 3. The results are shown in Figure 6, and it can be seen that the specific results meet the requirements.

[0096] Experimental Example 2 Linear test of the primer combination of the present invention

[0097] The characteristic genes of 12 foodborne pathogenic bacteria corresponding to the primer combination designed in Example 1 were cloned and constructed into plasmids, and were respectively diluted in a 10-fold gradient within the range of 10 - 10000 copies / ul for linearity testing. Using the primer combination of Example 1 of the present invention, according to the detection method of Example 3, templates diluted in 12 types of gradients were respectively detected. The positive one-dimensional scatter plot of digital PCR detection is as Figure 7 shown, and the linear analysis curve is as Figure 8 shown. The results show that R2 > 0.9999 for all template detections, indicating excellent linearity.

[0098] Experimental Example 3 Sample testing of the primer combination of the present invention

[0099] Using the detection methods described in Example 3 of the present invention and the existing detection methods respectively, 12 samples provided by Changhai Hospital were detected respectively. The results are shown in Table 9. It can be seen that in the clinical tests of 12 samples, the consistency between the method of the present invention and the culture method reached 100%.

[0100] Table 9

[0101]

[0102]

[0103] Experimental Example 4 Optimization research on the primers and probes of the present invention

[0104] During the design process of the primers and probes in Example 1 of the present invention, a series of different primer and probe combinations were tested to screen for primer and probe combinations that can achieve better technical effects. As shown in Tables 10 - 11, the primer and probe combination (highlighted) designed in Example 1 of the present invention has better technical effects compared to other available primer and probe combinations.

[0105] Table 10 Comparison of the effects of different primer and probe combinations - Primer combination A

[0106]

[0107]

[0108]

[0109] Table 11 Comparison of the effects of different primer and probe combinations - Primer combination B

[0110]

[0111]

[0112]

[0113] For example, two sets of primer-probes designed for Vibrio parahaemolyticus were tested using plasmids respectively. As Figure 9 shown, the signal-to-noise ratio of combination 2 is 5.07, which is higher than that of combination 1 (3.67). Therefore, combination 2 is preferred.

[0114] Two sets of primer-probes designed for Vibrio cholerae were tested using plasmids respectively. As Figure 10 shown, the amplification efficiency of combination 2 is higher, specifically manifested as fewer raindrops in the one-dimensional scatter plot and the basic consistency of the positive signal intensities of the two probes. Overall, combination 2 is preferred.

[0115] Two sets of primer-probes designed for Listeria monocytogenes were tested using plasmids respectively. As Figure 11 shown, the positive signal of combination 1 is higher, and the dispersion degree of positive points is lower (better and more consistent amplification efficiency). Overall, combination 1 is preferred.

[0116] Two sets of primer-probes designed for ipaH were tested using plasmids respectively. As Figure 12 shown, the positive signal of combination 2 is better, and the signal-to-noise ratio is higher (the signal-to-noise ratios of the two sets of primer-probes are 3.64 and 11.98 respectively). Overall, combination 2 is preferred.

[0117] Two sets of primer-probes designed for stx1 were tested using plasmids respectively. As Figure 13 shown, there is an abnormality in the amplification of combination 1, and the positive and negative droplets cannot be distinguished at all. Overall, combination 2 is preferred.

[0118] Two sets of primer-probes designed for rfbE were tested using plasmids respectively. As Figure 14 shown, the positive signal of combination 2 is higher, and the copy number is higher (1196 copies / μL and 2622.6 copies / μL respectively). Overall, combination 2 is preferred.

[0119] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A primer combination for multiplex detection of foodborne pathogenic bacteria, characterized in that, The primer combination includes: primer combination A and primer combination B; The primer combination A includes: probes and primer pairs for detecting 9 foodborne pathogenic bacteria; the 9 foodborne pathogenic bacteria include: Vibrio cholerae general type, Vibrio cholerae O1 type, Vibrio cholerae O139 type, Vibrio parahaemolyticus, Salmonella, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, and Enterobacter sakazakii; the probes and primer pairs in the primer combination A are as shown in the sequences: SEQ1 - SEQ31; The primer combination B includes: probes and primer pairs for detecting 9 characteristic genes of 3 foodborne pathogenic bacteria; the 9 characteristic genes include: Shigella genus general type: ipaH, diarrheagenic Escherichia coli general type: eltA, sta3, sta, aggR, enterohemorrhagic Escherichia coli: stx1, stx2, escV, rfbE; the probes and primer pairs in the primer combination B are as shown in the sequences: SEQ32 - SEQ64.

2. The primer combination according to claim 1, wherein The probe is labeled with a fluorescent group or a quenching group at the 5'-end and / or 3'-end.

3. The primer combination according to claim 2, wherein The fluorescent group is any one or more of FAM, HEX, ROX, CY5, Q705; the quenching group is any one or more of BHQ1, BHQ2, BHQ3.

4. A kit for multiplex detection of foodborne pathogenic bacteria, characterized in that, The kit includes the primer combination according to any one of claims 1 - 3.

5. The kit according to claim 4, wherein The kit further includes a detection Buffer, a detection enzyme, a positive control product, and a negative control product.

6. The kit according to claim 5, wherein The detection Buffer is dPCR Buffer; the detection enzyme is dPCR Taq enzyme; the positive control product is a mixed solution of artificial synthetic genes containing the foodborne pathogenic bacteria target sequence described in claim 1; the negative control product is nuclease-free water.

7. A detection method for multiplex detection of foodborne pathogenic bacteria, characterized in that, The detection method includes the following steps: (1) Extract the DNA of the sample to be tested; (2) Perform digital PCR reactions respectively using the primer combinations A and B according to any one of claims 1 - 3; (3) Combine the multi-color fluorescence decoding technology to perform genotyping detection on the results.

8. The detection method according to claim 7, characterized in that The primer combination includes primer combination A and primer combination B; when performing digital PCR reaction using primer combination A, the detection channels selected are FAM, HEX, ROX, and Cy5; when performing digital PCR reaction using primer combination B, the detection channels selected are FAM, HEX, ROX, Cy5, and Q705.

9. The detection method according to claim 7, wherein In the digital PCR reaction system, the final concentration of each probe in the primer combination is 100 - 400 nM, and the final concentration of each primer is 400 - 600 nM.

10. The detection method according to claim 7, characterized in that, The sample to be tested is a food sample or a human fecal sample.

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