Method for quantitatively detecting rice yeast acid virulent strain by using multiple digital PCR (Polymerase Chain Reaction) chip

Abstract

The invention relates to the technical field of food safety detection, in particular to a method for quantitatively detecting a rice yeast acid virulent strain by using a multiple digital PCR (Polymerase Chain Reaction) chip. The method mainly comprises the following steps: 1, preparing a chip, primers and a sample; 2, processing by using a micropump; 3, assembling the multiple digital PCR chip and the micropump; 4, distributing the primers, 5, drying and depositing the primers, 6, distributing the sample, 7, filling an oil phase, 8, carrying out PCR reaction, and 9, reading and analyzing a signal. The method has the advantages of accurate quantification, high specificity, high sensitivity, simple operation, low cost and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and particularly relates to a method for quantitatively detecting the strains producing bongkrekic acid by using a multiplex digital PCR chip. Background Art

[0002] Bongkrekic acid is a toxin produced by Pseudomonas cocovenenans. When people ingest food contaminated with this toxin, they may be poisoned, and in severe cases, it can lead to systemic organ failure, with a fatality rate as high as 40% - 100%. With the development of the economic society, people pay more and more attention to food safety, and the public's demand for the detection of bongkrekic acid is increasing day by day.

[0003] Currently, in the national standard GB 4789.29 - 2020 "Inspection of Burkholderia gladioli (Pseudomonas cocovenenans subsp. farinosa)" for the determination of bongkrekic acid in China, the detection methods of liquid - liquid extraction - thin - layer chromatography and liquid - liquid extraction - liquid chromatography are adopted. These methods have problems such as cumbersome and time - consuming sample pretreatment, many interferences, and the need to be equipped with expensive instrument equipment, resulting in high detection costs, long time consumption, and complex operations, and have great limitations in actual application. In the latest group standard T / SATA 023 - 2021 "Detection Method for Burkholderia gladioli and Strains Producing Bongkrekic Acid - Real - Time Fluorescent PCR Method", the qPCR detection method is adopted. The whole PCR process is monitored in real time by the accumulation of fluorescence signals, and the unknown template is quantified through a standard curve to achieve quantitative analysis of the unknown template. However, there are many factors affecting the amplification efficiency of qPCR, resulting in the cycle threshold (Ct), which is the basis of its quantitative analysis, not being constant. The quantification of qPCR is only "relative quantification", and in cases where the target sequence content is low, the expression level difference is very small, there are a large number of background sequences or inhibitors in the reaction system, etc., the sensitivity and accuracy are greatly limited.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above - mentioned deficiencies of the existing technology, the purpose of the present invention is to provide a method for quantitatively detecting the strains producing bongkrekic acid by using a multiplex digital PCR chip, aiming to solve the problem that the existing fluorescence quantitative PCR detection method has low detection sensitivity and accuracy.

[0006] Compared with fluorescence quantitative PCR, digital PCR does not need to rely on Ct values and standard curves. It can directly calculate the initial concentration of the target template in the sample based on the Poisson distribution principle, thus achieving absolute nucleic acid quantification. In addition, digital PCR can effectively avoid the influence of inhibitors in the reaction system, and the system distribution process also greatly reduces the concentration of background sequences that competitively interact with the target sequence. Therefore, it is particularly suitable for the detection of pathogens in complex matrices such as food. Currently, there are no reports on the related research using digital PCR to detect the strains producing bongkrekic acid.

[0007] The dispersion of nucleic acid samples is the first and most core step in digital PCR. There are mainly two ways of sample dispersion: droplet method and microfluidic chip method. In the droplet method, in order to maintain the dispersion of the decomposed droplets, surfactants need to be added. Such additives may have an impact or interference on some test systems, thus affecting the credibility of the results. Moreover, during the detection process, multiple droplet transfer operations and serial droplet signal readings are required, resulting in a relatively complex process and a long detection time. The microfluidic chip method is superior in detection accuracy and result accuracy, but the existing commercial PCR chip systems are complex and have high operating costs. Therefore, the present invention uses a multiplex digital PCR chip (for the structure and preparation method of the multiplex digital PCR chip, see Patent CN 107262170 A) to detect the strains producing bongkrekic acid. The multiplex digital PCR chip used in the present invention does not require the addition of surfactants, does not require precise micro-pump drive and complex micro-valve control, nor does it require complex macro-micro interfaces, and greatly reduces the complexity and operating costs of the digital PCR chip system, and can also improve the detection sensitivity and accuracy.

[0008] The technical solution of the present invention is as follows:

[0009] In the first aspect of the present invention, a method for quantitatively detecting the strains producing bongkrekic acid using a multiplex digital PCR chip is provided, which includes:

[0010] Providing a multiplex digital PCR chip;

[0011] Providing Burkholderia gladioli and the strains producing bongkrekic acid;

[0012] Providing primers for detecting Burkholderia gladioli and primers for detecting the strains producing bongkrekic acid;

[0013] Burkholderia gladioli:

[0014] Forward primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3'

[0015] Reverse primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3'

[0016] Bacillus cereus var. mycoides strain producing bongkrekic acid:

[0017] Forward primer: 5'-CGATGATATAGCCGAGGTT-3'

[0018] Reverse primer: 5'-CAGGTTCCAGTGCCATTA-3';

[0019] Extract nucleic acids from the Burkholderia gladioli and the Bacillus cereus var. mycoides strain producing bongkrekic acid respectively, and mix the extracted nucleic acids with polymerase, probe, and buffer to obtain a sample solution;

[0020] Add the primers for detecting Burkholderia gladioli and the primers for detecting the Bacillus cereus var. mycoides strain producing bongkrekic acid into the multiplex digital PCR chip, then add the sample solution, and after PCR reaction, read and analyze the fluorescence signals to obtain the content of the Bacillus cereus var. mycoides strain producing bongkrekic acid.

[0021] Optionally, the method specifically includes:

[0022] Step 1: Provide a multiplex digital PCR chip, which is composed of a top glass cover slip, a middle PDMS thin film, and a bottom glass substrate, and at least 2 microcavity array structures are arranged on the middle PDMS thin film;

[0023] Provide 2 PDMS micropumps with microcolumn array structures and 2 PDMS micropumps with through-hole structures;

[0024] Step 2: Provide Burkholderia gladioli and the Bacillus cereus var. mycoides strain producing bongkrekic acid;

[0025] Provide the primers and probes for detecting Burkholderia gladioli, and the primers and probes for detecting the Bacillus cereus var. mycoides strain producing bongkrekic acid;

[0026] Burkholderia gladioli:

[0027] Forward primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3'

[0028] Reverse primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3'

[0029] Probe:

[0030] 5'-FAM-ATTGTTAAAGAACGACAGCCGATAAGCACAC-BHQ-3'

[0031] Bacillus cereus var. mycoides strain producing bongkrekic acid:

[0032] Forward primer: 5'-CGATGATATAGCCGAGGTT-3'

[0033] Reverse primer: 5'-CAGGTTCCAGTGCCATTA-3'

[0034] Probe: 5'-FAM-CGATGGTCCGTATCTCCTGCTTGTGC-BHQ-3';

[0035] Step 3: Extract nucleic acids from Burkholderia gladioli and the strain producing bongkrekic acid respectively, and mix the extracted nucleic acids with polymerase, probe, and buffer to obtain sample solutions;

[0036] Step 4: Place 2 PDMS micropumps with microcolumn array structures and 2 PDMS micropumps with through-hole structures in a vacuum container for degassing treatment for at least 60 minutes, and then vacuum seal them for standby;

[0037] Step 5: Bond the treated PDMS micropump with a microcolumn array structure to the bottom glass substrate of the multiplex digital PCR chip, and then bond the treated PDMS micropump with a through-hole structure to the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, so that the through holes of the PDMS micropump with a through-hole structure communicate with the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, and the unbonded through-hole surface is sealed with tape;

[0038] Step 6: Drop the 2 primers in Step 2 into the 2 primer inlets II on the top glass cover of the multiplex digital PCR chip after being treated in Step 5 respectively. The 2 primers will be driven by negative pressure and enter the respective microcavities in the microcavity array structure corresponding to each primer inlet II on the middle PDMS film until the excess primers are discharged from the waste liquid outlet II on the top glass cover;

[0039] Step 7: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip after being treated in Step 6. Then seal the 2 primer inlets II on the top glass cover of the multiplex digital PCR chip with tape and perform freeze-drying treatment on the multiplex digital PCR chip to deposit the primers in each microcavity at the bottom of the microcavity;

[0040] Step 8: Take out the multiplex digital PCR chip after being treated in Step 7, repeat the treatment method in Step 5, and then drop the sample solution prepared in Step 3 into the sample inlet II on the top glass cover of the multiplex digital PCR chip. The sample solution will be driven by negative pressure and enter the respective microcavities in the microcavity array structure on the middle PDMS film until the excess sample solution is discharged from the waste liquid outlet II on the top glass cover;

[0041] Step 9: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip after being processed in Step 8, and drop the oil phase at the sample injection port II, primer injection port II, and waste liquid outlet II on the top glass cover slip of the multiplex digital PCR chip and at the inlet and outlet on the bottom glass substrate respectively.

[0042] Step 10: After sealing the sample injection port II, primer injection port II, and waste liquid outlet II on the top glass cover slip of the multiplex digital PCR chip and the inlet and outlet on the bottom glass substrate that have been processed in Step 9 with tape, place it on an in-situ PCR instrument for thermal cycle amplification reaction.

[0043] Step 11: Read and analyze the fluorescence signal of the multiplex digital PCR chip after being processed in Step 10 through a fluorescence microscope or scanner to obtain the contents of Burkholderia gladioli and the strains producing bongkrekic acid in the sample to be tested.

[0044] Optionally, the 3' ends of the probes for detecting Burkholderia gladioli and the 3' ends of the probes for detecting the strains producing bongkrekic acid in Step 2 are both labeled with a fluorescence quenching group such as one of BHQ, TAMRA, Eclipse, Dabcy, and Lowa Black TM RQ.

[0045] The 5' ends of the probes for detecting Burkholderia gladioli and the 5' ends of the probes for detecting the strains producing bongkrekic acid are both labeled with a fluorescence reporting group such as one of FAM, JOE, TET, HEX, VIC, CY5, and CY3.

[0046] Optionally, in Step 3, nucleic acid is extracted from Burkholderia gladioli by the boiling method, and nucleic acid is extracted from the strains producing bongkrekic acid by the boiling method.

[0047] Optionally, the polymerase in Step 3 is Premix Ex TaqTM (Probe qPCR; TaKaRa, Tokyo, Japan).

[0048] Optionally, the PCR reaction system in each microchamber in Step 8 includes 6 μL of Premix Ex Taq TM (probe qPCR), 0.6 μL of upstream primer (10 nM), 0.6 μL of downstream primer (10 nM), 0.24 μL of fluorescence probe (10 nM), 0.6 μL of BSA solution (10 mg / mL), 12.76 μL of nuclease-free water, and 1.2 μL of DNA template solution (i.e., nucleic acid solution).

[0049] Optionally, the oil phase in Step 9 is silicone oil.

[0050] Optionally, the in-situ PCR instrument described in step 10 is Eppendorf (from Hamburg, Germany); the thermal cycling amplification reaction conditions are: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 40 s, annealing and extension at 56°C for 40 s, for a total of 45 cycles; incubation at 4°C.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] It is possible to simultaneously quantitatively detect Burkholderia gladioli and the toxin-producing strain of bongkrekic acid without enrichment, and the detection limit can reach 5 fg / μL of DNA template and CFU / 25 mL of bacterial solution;

[0053] Compared with fluorescence quantitative PCR, the multiplex digital PCR chip method of the present invention does not need to rely on Ct values and standard curves, and can directly rely on the Poisson distribution principle to calculate the initial concentration of the target template in the sample, thereby realizing absolute nucleic acid quantification. The detection method of the present invention also has the advantages of high sensitivity, good specificity and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a fluorescence microscopic image of the multiplex digital PCR chip of the present invention;

[0055] Figure 2 It is the amplification result diagram of the multiplex digital PCR chip detection method of the present invention for Burkholderia gladioli and the toxin-producing strain of bongkrekic acid; among them, (a) is the negative control, which is the fluorescence microscopic image in the case of no target DNA template; (b)-(f) are the fluorescence microscopic images of the serial dilutions containing the target DNA template, with concentrations ranging from 5 copies / μL to 5×10 4 copies / μL;

[0056] Figure 3 It is the linear relationship diagram between the concentration (copies / μL) measured by dPCR and the expected concentration (copies / μL). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0057] The following will describe the implementation scheme of the present invention in detail in combination with the embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0058] The embodiment of the present invention provides a method for quantitatively detecting the toxin-producing strain of bongkrekic acid using a multiplex digital PCR chip, which includes:

[0059] Provide a multiplex digital PCR chip;

[0060] Provide Burkholderia gladioli and the toxin-producing strain of bongkrekic acid;

[0061] Provide primers for detecting Burkholderia gladioli and primers for detecting the toxin-producing strain of bongkrekic acid;

[0062] Burkholderia gladioli:

[0063] Forward primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3'

[0064] Reverse primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3'

[0065] Toxin-producing strain of bongkrekic acid:

[0066] Forward primer: 5'-CGATGATATAGCCGAGGTT-3'

[0067] Reverse primer: 5'-CAGGTTCCAGTGCCATTA-3;

[0068] Extract nucleic acids from Burkholderia gladioli and the toxin-producing strain of bongkrekic acid respectively, and mix the extracted nucleic acids with polymerase, probe, and buffer to obtain a sample solution;

[0069] Add the primers for detecting Burkholderia gladioli and the primers for detecting the toxin-producing strain of bongkrekic acid into the multiplex digital PCR chip, then add the sample solution. After PCR reaction, read and analyze the fluorescence signal to obtain the content of the toxin-producing strain of bongkrekic acid.

[0070] In one embodiment, the method for quantitatively detecting the toxin-producing strain of bongkrekic acid using a multiplex digital PCR chip specifically includes:

[0071] Step 1: Provide a multiplex digital PCR chip, which is composed of a top glass cover slip, a middle PDMS thin film, and a bottom glass substrate. At least 2 microcavity array structures are provided on the middle PDMS thin film;

[0072] Provide 2 PDMS micropumps with microcolumn array structures and 2 PDMS micropumps with through-hole structures;

[0073] Step 2: Provide Burkholderia gladioli and the toxin-producing strain of bongkrekic acid;

[0074] Provide primers and probes for detecting Burkholderia gladioli and primers and probes for detecting the toxin-producing strain of bongkrekic acid;

[0075] Burkholderia gladioli:

[0076] Forward primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3'

[0077] Reverse primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3'

[0078] Probe:

[0079] 5'-FAM-ATTGTTAAAGAACGACAGCCGATAAGCACAC-BHQ-3'

[0080] Bongkrekic acid-producing strain:

[0081] Forward primer: 5'-CGATGATATAGCCGAGGTT-3'

[0082] Reverse primer: 5'-CAGGTTCCAGTGCCATTA-3'

[0083] Probe: 5'-FAM-CGATGGTCCGTATCTCCTGCTTGTGC-BHQ-3;

[0084] Step 3: Extract nucleic acids from Burkholderia gladioli and the bongkrekic acid-producing strain respectively, and mix the extracted nucleic acids with polymerase, probe, and buffer to obtain sample solutions;

[0085] Step 4: Place 2 PDMS micropumps with microcolumn array structures and 2 PDMS micropumps with through-hole structures in a vacuum container for at least 60 minutes of degassing treatment, and vacuum seal them for standby respectively;

[0086] Step 5: Bond the treated PDMS micropump with a microcolumn array structure to the bottom glass substrate of the multiplex digital PCR chip, and then bond the treated PDMS micropump with a through-hole structure to the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, so that the through holes of the PDMS micropump with a through-hole structure communicate with the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, and the unbonded through-hole surface is sealed with tape;

[0087] Step 6: Drop the 2 primers in Step 2 into the 2 primer inlets II on the top glass cover of the multiplex digital PCR chip treated in Step 5 respectively. The 2 primers will be driven by negative pressure and enter the respective microcavities in the microcavity array structure corresponding to each primer inlet II on the middle PDMS film until the excess primers are discharged from the waste liquid outlet II on the top glass cover;

[0088] Step 7: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip after being processed in Step 6. Then, seal the two primer inlets II on the top glass cover slip of the multiplex digital PCR chip with tape, and perform freeze-drying treatment on the multiplex digital PCR chip to deposit the primers in each microcavity at the bottom of the microcavity.

[0089] Step 8: Take out the multiplex digital PCR chip after being processed in Step 7, repeat the processing method of Step 5, and then drop the sample solution prepared in Step 3 at the sample inlet II on the top glass cover slip of the multiplex digital PCR chip. The sample solution is driven by negative pressure into each microcavity in the microcavity array structure on the middle PDMS film until the excess sample solution is discharged from the waste liquid outlet II on the top glass cover slip.

[0090] Step 9: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip after being processed in Step 8, and drop oil phases at the sample inlet II, primer inlet II, and waste liquid outlet II on the top glass cover slip of the multiplex digital PCR chip and at the inlet and outlet on the bottom glass substrate respectively.

[0091] Step 10: After sealing the sample inlet II, primer inlet II, and waste liquid outlet II on the top glass cover slip of the multiplex digital PCR chip and the inlet and outlet on the bottom glass substrate with tape after being processed in Step 9, place it on an in-situ PCR instrument for thermal cycle amplification reaction.

[0092] Step 11: Read and analyze the fluorescence signals of the multiplex digital PCR chip after being processed in Step 10 through a fluorescence microscope or scanner to obtain the contents of Burkholderia gladioli and the strains producing bongkrekic acid in the sample to be tested.

[0093] The present invention can repeat this method using various sample solutions with different concentrations for detection to verify the accuracy of the detection method results.

[0094] The present invention can use the preserved Burkholderia gladioli and the strains producing bongkrekic acid to contaminate rice noodles respectively. Using the contaminated rice noodles as samples, extract nucleic acids respectively and then perform detection with reference to the above method. After verifying the accuracy of the results, apply them to the detection of food samples.

[0095] It can simultaneously quantitatively detect Burkholderia gladioli and the strains producing bongkrekic acid without enrichment, and the detection limit can reach 5 fg / μL of DNA template and CFU / 25 mL of bacterial solution.

[0096] Compared with fluorescence quantitative PCR, the multiplex digital PCR chip method of the present invention does not need to rely on Ct values and standard curves, and can directly rely on the Poisson distribution principle to calculate the initial concentration of the target template in the sample, so as to achieve absolute nucleic acid quantification. The verification results show that the detection method of the present invention has the advantages of high sensitivity, good specificity and high precision.

[0097] Among them, the multiplex digital PCR chip in step 1 can be prepared by the method disclosed in Patent CN107262170 A.

[0098] Among them, the probe for detecting Burkholderia gladioli in step 2 is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end. The fluorescent group can be selected from any one of the fluorescent reporter groups such as FAM, JOE, TET, HEX, VIC, CY5, CY3, and preferably FAM;

[0099] The quenching group can be selected from any one of the fluorescent quenching groups such as BHQ, TAMRA, Eclipse, Dabcy, Lowa Black TM RQ, and preferably BHQ.

[0100] Among them, the probe for detecting the strain producing bongkrekic acid in step 2 is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end. The fluorescent group can be selected from any one of the fluorescent reporter groups such as FAM, JOE, TET, HEX, VIC, CY5, CY3, and preferably FAM;

[0101] The quenching group can be selected from any one of the fluorescent quenching groups such as BHQ, TAMRA, Eclipse, Dabcy, Lowa Black TM RQ, and preferably BHQ.

[0102] The present invention will be further described in detail below through specific examples.

[0103] First, it should be noted that for the following examples, those without specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0104] Examples

[0105] I. Main strains

[0106] The strains used in the examples are: Burkholderia gladioli and the strain producing bongkrekic acid. II. Main detection equipment used

[0107] Multiplex digital PCR chip

[0108] In-situ PCR instrument

[0109] Fluorescence microscope for reading fluorescence signals

[0110] III. Main reagents used

[0111] DNA extraction reagent

[0112] IV. A method for quantitatively detecting Burkholderia gladioli strains producing bongkrekic acid using a multiplex digital PCR chip, the method comprising the following steps:

[0113] 1. Fabricate the chip and micropump: Fabricate multiple multiplex digital PCR chips each containing 2 microcavity arrays, PDMS micropumps with microcolumn array structures, and PDMS micropumps with through-hole structures respectively according to the method disclosed in Patent CN107262170 A;

[0114] 2. Purchase specific primers and probes for Burkholderia gladioli and Burkholderia gladioli strains producing bongkrekic acid respectively: As shown below, the 5' end of the probe is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ;

[0115] Burkholderia gladioli:

[0116] Forward primer (Gladiolus - PBAF): 5'-GCTTCCGCTATCCAAATTACTACTTC-3'

[0117] Reverse primer (Gladiolus - PBAR): 5'-ATGACAAATGTTCGAGTCAGTTGAC-3'

[0118] Probe (Gladiolus - PBA - FAM):

[0119] 5'-FAM-ATTGTTAAAGAACGACAGCCGATAAGCACAC-BHQ-3'

[0120] Burkholderia gladioli strains producing bongkrekic acid:

[0121] Forward primer (BA1F): 5'-CGATGATATAGCCGAGGTT-3'

[0122] Reverse primer (BA1R): 5'-CAGGTTCCAGTGCCATTA-3'

[0123] Probe (BA1 - FAM):

[0124] 5'-FAM-CGATGGTCCGTATCTCCTGCTTGTGC-BHQ-3;

[0125] 3. Sample preparation: Extract nucleic acids from the preserved Burkholderia gladioli and the strain producing bongkrekic acid respectively. Specifically, the extraction steps are as follows: Take 1 mL of the bacterial solution and add it to a 1.5 mL centrifuge tube. Centrifuge at 12,000 g for 2 minutes, and aspirate the supernatant. Add 500 μL of sterile water, mix well and centrifuge at 12,000 g for 2 minutes, then aspirate the supernatant. Add 100 μL of sterile water, heat in a boiling water bath for 10 minutes, centrifuge at 12,000 g for 2 minutes, and take the supernatant and store it at -20 °C for later use. Mix the extracted nucleic acids with polymerase, probe, buffer, etc. respectively to prepare sample solutions with known concentrations. Among them, the polymerase is Premix Ex TaqTM (Probe qPCR; TaKaRa, Tokyo, Japan).

[0126] 4. Micropump treatment: Place 2 PDMS micropumps with microcolumn array structures and 2 PDMS micropumps with through-hole structures in a vacuum container for at least 60 minutes of degassing treatment, and then vacuum seal them for later use respectively.

[0127] 5. Assembly of the multiplex digital PCR chip and the micropump: Attach the processed PDMS micropump with a microcolumn array structure to the bottom glass substrate of the multiplex digital PCR chip, and then attach the processed PDMS micropump with a through-hole structure to the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, so that the through holes of the PDMS micropump with a through-hole structure communicate with the waste liquid outlet II on the top glass cover of the multiplex digital PCR chip, and the unbonded through-hole surface is sealed with tape.

[0128] 6. Primer dispensing: Drop the 2 primers in step 2 into the 2 primer inlets II on the top glass cover of the multiplex digital PCR chip processed in step 5 respectively. Driven by negative pressure, the 2 primers will enter the respective microcavities in the microcavity array structure corresponding to each primer inlet II on the middle PDMS film until the excess primers are discharged from the waste liquid outlet II on the top glass cover to ensure that each primer is independently dispensed into each microcavity.

[0129] 7. Primer drying and deposition treatment: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip processed in step 6. Then seal the 2 primer inlets II on the top glass cover of the multiplex digital PCR chip with tape and perform freeze-drying treatment on the multiplex digital PCR chip to deposit the primers in each microcavity at the bottom of the microcavity.

[0130] 8. Sample dispensing: Take out the multiplex digital PCR chip processed in step 7, repeat the processing method of step 5, and then add the sample solution prepared in step 3 dropwise to the sample injection port II on the top glass cover of the multiplex digital PCR chip. The sample solution is driven by negative pressure to enter each microcavity in the microcavity array structure on the middle PDMS film until the excess sample solution is discharged from the waste liquid outlet II on the top glass cover to ensure that each sample solution is independently dispensed into each microcavity. The sample solution entering each microcavity will dissolve the primer deposited at the bottom of each microcavity to form a complete PCR reaction system;

[0131] Among them, the PCR reaction system in each microcavity includes 6 μL of Premix Ex Taq TM (probe qPCR), 0.6 μL of upstream primer (10 nM), 0.6 μL of downstream primer (10 nM), 0.24 μL of fluorescent probe (10 nM), 0.6 μL of BSA solution (10 mg / mL), 12.76 μL of nuclease-free water, and 1.2 μL of DNA template solution (i.e., nucleic acid solution);

[0132] 9. Filling the oil phase: Peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip processed in step 8, and add the oil phase (the oil phase is silicone oil) dropwise to the sample injection port II, primer injection port II, and waste liquid outlet II on the top glass cover of the multiplex digital PCR chip and the inlet and outlet on the bottom glass substrate respectively. The oil phase fills the other spaces except the microcavity array structure in the closed micro-pipeline system formed by the bonding of the top glass cover and the middle PDMS film and the closed cavity formed by the bonding of the bottom glass substrate and the middle PDMS film through capillary action; respectively realize the isolation of the reaction systems in each microcavity in the microcavity array structure on the middle PDMS film and prevent the rapid evaporation of the droplet moisture in the microcavity during the PCR reaction;

[0133] 10. PCR reaction: After sealing the sample injection port II, primer injection port II, and waste liquid outlet II on the top glass cover of the multiplex digital PCR chip processed in step 9 and the inlet and outlet on the bottom glass substrate with tape, place it on an in-situ PCR instrument for thermal cycle amplification reaction;

[0134] Among them, the in-situ PCR instrument is Eppendorf; the thermal cycle amplification reaction conditions are: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 40 s, annealing and extension at 56 °C for 40 s, for a total of 45 cycles; incubation at 4 °C;

[0135] 11. Signal reading and analysis: Fluorescence signals of the multiplex digital PCR chip after being processed in step 10 are read and analyzed by a fluorescence microscope to analyze the contents of Burkholderia gladioli and the strains producing bongkrekic acid in the sample to be tested, and verify the accuracy of the detection results.

[0136] 12. Result verification: The detection method is repeated using sample solutions with various different concentrations to verify the accuracy of the results of the detection method.

[0137] 13. Application: Burkholderia gladioli and the strains producing bongkrekic acid that have been preserved are used to contaminate rice noodles respectively. Taking the contaminated rice noodles as samples, nucleic acids are extracted respectively and then detected according to the above method. After verifying the accuracy of the results, it is applied to the detection of food samples.

[0138] 14. Specificity evaluation of the multiplex digital PCR chip for detecting the strains producing bongkrekic acid

[0139] The multiplex digital PCR chip is used to detect Burkholderia gladioli and the strains producing bongkrekic acid respectively with the primer-probe of the present invention to verify the specificity of the method.

[0140] Figure 1 are two fluorescence microscopic images of the multiplex digital PCR chip. It can be seen from the experiment that the chambers without the primer-probe of the present invention show low fluorescence levels (i.e., "negative signals"), while the chambers with the primer-probe of the present invention show high fluorescence levels (i.e., "positive signals"). This result indicates that the primers and probes of this multiplex digital PCR chip system have good specificity and can be used for subsequent experiments. The primers, probes and droplet digital PCR method established by the present invention can only specifically amplify Salmonella.

[0141] 15. Sensitivity evaluation of the multiplex digital PCR chip for detecting the strains producing bongkrekic acid

[0142] The DNA solution of the strains producing bongkrekic acid with a concentration of 50 ng / μL is serially diluted according to a 10-fold concentration gradient to measure the concentration of the initial DNA solution. The initial DNA solution is continuously diluted with Tris-EDTA buffer to obtain DNA solutions of 5 copies / μL, 50 copies / μL, 500 copies / μL, 5000 copies / μL and 50000 copies / μL. Their amplification results are as Figure 2 shown in (a)-(f). The sensitivity of the multiplex digital PCR chip is evaluated using the primer-probe of the present invention. Figure 3 is the linear relationship between the dPCR measured concentration (copies / μL) and the expected concentration (copies / μL). The data are presented as the mean value, and all experiments are repeated three times. The linear relationship is y = 0.97884x + 0.07113 (R 2= 0.9984), the concentration of the original template was 1.95768×10 5 copies / μL, and the actual concentration was 2×10 5 copies / μL. The experimental results were in good agreement with the expected values, proving that the dPCR chip had high sensitivity and accuracy. The generated quantity reached the experimental requirements. As the concentration of the template DNA increased, the number of positives increased. When the template concentration was as low as 10 -8 , positive results could still be observed, indicating that the sensitivity of the multiplex digital PCR chip method for genomic DNA could reach 5 fg / μL.

[0143] 16. Detection of artificially contaminated rice noodle samples using a multiplex digital PCR chip

[0144] The preserved Burkholderia gladioli and the strain producing bongkrekic acid were used to contaminate rice noodles respectively. The contaminated rice noodles were used as samples, and nucleic acids were extracted respectively and then detected according to the above method. The rice noodle samples without artificial contamination were used as the control group.

[0145] The amplification results were as Figure 2 shown. When using the multiplex digital PCR chip of the present invention to detect artificially contaminated rice noodles, its sensitivity could reach CFU / 25 mL.

[0146] In summary, the present invention provides a method for quantitatively detecting the strain producing bongkrekic acid using a multiplex digital PCR chip. The multiplex digital PCR chip method of the present invention can simultaneously quantitatively detect Burkholderia gladioli and the strain producing bongkrekic acid without enrichment, and the detection limit can reach a DNA template of 5 fg / μL and a bacterial liquid of CFU / 25 mL; compared with fluorescence quantitative PCR, the multiplex digital PCR chip method of the present invention does not need to rely on Ct values and standard curves, and can directly rely on the Poisson distribution principle to calculate the initial concentration of the target template in the sample, so as to achieve absolute nucleic acid quantification. The detection method of the present invention also has the advantages of high sensitivity, good specificity and high precision.

[0147] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all these improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip, characterized in that: include: Provide multiplex digital PCR chips; Provide Burkholderia gladiolus and fumonisin-producing strains; Provide primers for the detection of Burkholderia gladiolus and primers for the detection of fumonisin-producing strains; Burkholderia gladiolus: Upstream primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3' Downstream primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3' Bacillus flavus-producing strains: Upstream primer: 5'-CGATGATATAGCCGAGGTT-3' Downstream primer: 5′-CAGGTTCCAGTGCCATTA-3′; Extracting nucleic acids from the gladiolus Burkholderia and the fumonisin-producing strain respectively, and mixing the extracted nucleic acids with a polymerase, a probe, and a buffer to obtain a sample solution; Primers for detecting Burkholderia gladiolus and primers for detecting fumonisin-producing strains are added to the multiplex digital PCR chip, and then a sample solution is added. After a PCR reaction, the fluorescence signal is read and analyzed to obtain the content of fumonisin-producing strains.

2. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 1, characterized in that: The method specifically comprises: Step 1, providing a multiplex digital PCR chip, wherein the multiplex digital PCR chip is composed of a top glass cover, a middle PDMS film and a bottom glass substrate, and at least two microcavity array structures are arranged on the middle PDMS film; Provide 2 PDMS micropumps with micro-pillar array structures and 2 PDMS micropumps with through-hole structures; Step 2, providing Burkholderia gladiolus and fumonisin-producing strains; Provide primers and probes for the detection of Burkholderia gladiolus and primers and probes for the detection of fumonisin-producing strains; Burkholderia gladiolus: Upstream primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3' Downstream primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3' Probe: 5'-FAM-ATTGTTAAAGAACGACAGCCGATAAGCACAC-BHQ-3' Bacillus flavus-producing strains: Upstream primer: 5'-CGATGATATAGCCGAGGTT-3' Downstream primer: 5'-CAGGTTCCAGTGCCATTA-3' Probe: 5′-FAM-CGATGGTCCGTATCTCCTGCTTGTGC-BHQ-3′; Step 3, respectively extracting nucleic acids from Burkholderia gladiolus and the fumonisin-producing strain, and respectively mixing the extracted nucleic acids with a polymerase, a probe, and a buffer to obtain a sample solution; Step 4, placing two PDMS micropumps with microcolumn array structures and two PDMS micropumps with through-hole structures in a vacuum container for at least 60 minutes for degassing, and vacuum-packaging them for standby use; Step 5, laminating the treated PDMS micropump with the microcolumn array structure to the bottom glass substrate of the multiple digital PCR chip, and then laminating the treated PDMS micropump with the through-hole structure to the waste liquid outlet II on the top glass cover of the multiple digital PCR chip, so that the through-hole of the PDMS micropump with the through-hole structure is connected to the waste liquid outlet II on the top glass cover of the multiple digital PCR chip, and the unlaminated through-hole surface is sealed with tape; Step 6, respectively dripping the two primers in step 2 into the two primer injection ports II on the top glass cover of the multiplex digital PCR chip treated in step 5, and the two primers are driven by negative pressure to enter each microcavity in the microcavity array structure corresponding to each primer injection port II on the middle PDMS film, until the excess primers are discharged from the waste liquid outlet II on the top glass cover; Step 7, peeling off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip processed in step 6, and then sealing the two primer injection ports II on the top glass cover of the multiplex digital PCR chip with tape, and then freeze-drying the multiplex digital PCR chip, so that the primers in each microcavity are deposited at the bottom of the microcavity; Step 8, taking out the multiplex digital PCR chip treated in step 7, repeating the treatment method of step 5, and then dripping the sample solution prepared in step 3 into the sample inlet II on the top glass cover of the multiplex digital PCR chip, and the sample solution is driven by negative pressure into each microcavity in the microcavity array structure on the middle PDMS film until the excess sample solution is discharged from the waste liquid outlet II on the top glass cover; Step 9, peel off the PDMS micropump with a microcolumn array structure and the PDMS micropump with a through-hole structure on the multiplex digital PCR chip processed in step 8, and drip oil phase into the sample inlet II, primer inlet II and waste liquid outlet II on the top glass cover of the multiplex digital PCR chip and the inlet and outlet on the bottom glass substrate respectively; Step 10, after sealing the sample inlet II, primer inlet II and waste liquid outlet II on the top glass cover sheet of the multiplex digital PCR chip processed in step 9 and the inlet and outlet on the bottom glass substrate with tape, place them on an in-situ PCR instrument for thermal cycle amplification reaction; Step 11: read and analyze the fluorescence signal of the multiplex digital PCR chip after the treatment in step 10 by using a fluorescence microscope or scanner to obtain the content of Burkholderia gladiolus and fumonisin-producing strains in the sample to be tested.

3. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: The 3' end of the probe for detecting Burkholderia gladioli and the 3' end of the probe for detecting fumonisin-producing strains in step 2 are both labeled with a fluorescence quenching group selected from BHQ, TAMRA, Eclipse, Dabcy, and Lowa Black TM RQ. The 5' end of the probe for detecting Burkholderia gladiolus and the 5' end of the probe for detecting fumonisin-producing strains are both labeled with a fluorescent reporter group selected from FAM, JOE, TET, HEX, VIC, CY5 and CY3.

4. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: In step 3, the nucleic acid is extracted from Burkholderia gladiolus by boiling method, and the nucleic acid is extracted from the fumonisin-producing strain by boiling method.

5. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: The polymerase in step 3 is Premix Ex Taq TM .

6. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: The PCR reaction system in each microchamber in step 8 includes 6 μL of Premix Ex Taq TM , 0.6 μL upstream primer, 0.6 μL downstream primer, 0.24 μL fluorescent probe, 0.6 μL BSA solution, 12.76 μL nuclease-free water and 1.2 μL nucleic acid solution.

7. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: The oil phase in step 9 is silicone oil.

8. The method for quantitatively detecting fumonisin-producing strains using a multiplex digital PCR chip according to claim 2, characterized in that: The in situ PCR instrument in step 10 is Eppendorf; the thermal cycle amplification reaction conditions are: 95°C pre-denaturation for 30s; 95°C denaturation for 40s, 56°C annealing and extension for 40s, a total of 45 cycles; 4°C insulation.

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