Dual digital PCR (Polymerase Chain Reaction) detection method for monkey campylobacter jejuni and monkey enterocolitis yersinia

Abstract

The invention discloses a dual digital PCR (polymerase chain reaction) detection method for monkey campylobacter jejuni and monkey enterocolitis yersinia, and relates to the technical field of microbiological detection, the detection method comprises the following specific steps: multi-source heterogeneous data collection: collecting monkey feces and intestinal tissue samples, extracting genome DNA (deoxyribonucleic acid) by using a phenol-chloroform method, detecting the concentration and purity of DNA, and detecting the concentration and purity of the DNA; after reaching the standard, storing at-20 DEG C for later use; according to the method, high-sensitivity and specific detection on the two pathogenic bacteria is realized through a combination of well-designed primers and probes, according to the method, a gyrA gene conserved region of monkey campylobacter jejuni CJ and a POL gene conserved region of monkey yersinia enterocolitica YE are used as detection targets, and specific fragments of 249bp and 182bp are amplified respectively; the method effectively avoids interference of non-specific amplification, can simultaneously detect two pathogenic bacteria in a single reaction system through a dual digital PCR technology, accurately calculates target gene copy number concentration through a Poisson distribution formula, and improves detection accuracy and reliability.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica. Background Art

[0002] Campylobacter jejuni (CJ) and Yersinia enterocolitica (YE) are two important animal-borne pathogens. They have a high infection rate in primates, especially non-human primates, and can cause severe gastrointestinal diseases and even death. With the deepening of primate experimental and conservation research worldwide, the rapid and accurate detection of these two pathogens has become particularly important. Traditional pathogen detection methods, such as bacterial culture and serological testing, although they can meet the detection needs to a certain extent, have problems such as complex operation, long time consumption and low sensitivity, which cannot meet the needs of rapid diagnosis and scientific research. Therefore, the development of an efficient, sensitive and specific dual detection method is of great significance for primate health monitoring, disease prevention and control, and related scientific research.

[0003] Traditional pathogen detection technology has many limitations. First, although bacterial culture methods can directly observe the growth of pathogens, the culture cycle is long, and some pathogens are difficult to grow under conventional culture conditions, resulting in a high missed detection rate. Second, serological detection methods rely on antigen-antibody reactions, which have problems of cross-reaction and non-specific binding, which may affect the accuracy of the test results. In addition, although traditional PCR technology has improved the sensitivity of detection to a certain extent, it is easily affected by PCR inhibitors and it is difficult to achieve simultaneous detection of multiple pathogens, which limits its application in complex samples. Therefore, traditional technologies are insufficient in detection sensitivity, specificity, ease of operation and multiple detection capabilities, and it is difficult to meet the needs of modern pathogen detection.

[0004] Therefore, the development of a dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica can not only be used for the rapid diagnosis of monkey Campylobacter jejuni and monkey Yersinia enterocolitica in clinical samples, providing strong support for the early detection and treatment of the disease; it can also be applied to primate protection research, disease monitoring and public health and safety, helping to timely detect and control the spread of pathogens and ensure the health and safety of primates. Summary of the Invention

[0005] The purpose of the present invention is to make up for the shortcomings of the existing technology and provide a dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica. This method collects monkey feces and intestinal tissue samples, extracts genomic DNA, and designs primers and probes based on the specific genes of the two pathogens to construct a dual digital PCR reaction system. Through droplet generation and distribution, digital PCR amplification and fluorescence signal detection, high-sensitivity and specific dual detection of the two pathogens is achieved.

[0006] To solve the above technical problems, the present invention provides the following technical solution: a dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica, the specific steps of the detection method are:

[0007] S100, multi-source heterogeneous data acquisition: monkey feces and intestinal tissue samples were collected, genomic DNA was extracted using the phenol-chloroform method, DNA concentration and purity were tested, and after reaching the standard, it was stored at -20°C for future use;

[0008] S200, primer and probe design and synthesis: Primers and probes were designed based on the conserved regions of the gyrA gene of monkey Campylobacter jejuni CJ and the POL gene of monkey Yersinia enterocolitica YE. The CJ primer-probe set amplified a 249 bp fragment, and the YE primer-probe set amplified a 182 bp fragment. The primers and probes were prepared into a 10 μM storage solution and stored at -20°C.

[0009] S300, preparation of duplex digital PCR reaction system: prepare 20 μL of system on ice, containing 10 μL 2× digital PCR premix, 2 μL DNA template, 0.6 μL each of CJ and YE upstream and downstream primers, and 0.2 μL each of CJ and YE probes. Make up with nuclease-free pure water, mix well, and centrifuge;

[0010] S400, droplet generation and distribution: The reaction solution and droplet generation oil are added to the droplet generation chip at a ratio of 1:2. The reaction system is added to the sample well, 70 μL of digital PCR oil is added to the oil phase well, and the chip is placed in the droplet generator to generate water-in-oil droplets.

[0011] S500, digital PCR amplification program: transfer the droplet to a PCR tube, pre-denaturation at 95°C for 15 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, 40 cycles, final denaturation at 98°C for 10 minutes, and then hold at 20°C for 2 minutes, with a temperature ramp rate of 2.0°C / s;

[0012] S600, fluorescence signal detection and data analysis: A fluorescence scanner is used to detect FAM and VIC fluorescence signals, the number of positive droplets is counted, and the target gene copy number concentration is calculated based on the Poisson distribution. A positive result is determined if there are ≥3 positive droplets in the CJ or YE channel and the concentration is ≥3.0 copies / μL; otherwise, it is considered negative.

[0013] Furthermore, the specific steps of extracting sample genomic DNA using the phenol-chloroform method are:

[0014] (1) Place the collected feces and intestinal tissue samples in a mortar and add liquid nitrogen to grind them thoroughly until they become powder;

[0015] (2) Transfer the ground powder to a centrifuge tube, add lysis buffer containing SDS and proteinase K, mix gently, and incubate in a 56°C water bath for 1-2 hours to lyse the cells and release genomic DNA;

[0016] (3) Add an equal volume of phenol-chloroform-isoamyl alcohol mixture to the centrifuge tube and mix by inversion for 10-15 minutes to denature the protein and separate the nucleic acid;

[0017] (4) Centrifuge at 12000 rpm for 10 minutes at room temperature. After the solution is separated, transfer the upper aqueous phase to a new centrifuge tube.

[0018] (5) Repeat steps (3)-(4) 1-2 times until there is no obvious protein precipitation at the interface after centrifugation;

[0019] (6) Add 0.1 volumes of 3 M sodium acetate and 2 volumes of anhydrous ethanol to the supernatant, mix thoroughly by inversion, and precipitate in a -20°C refrigerator for 1-2 hours;

[0020] (7) Centrifuge at 12000 rpm for 15 minutes, discard the supernatant, and retain the DNA pellet at the bottom of the tube;

[0021] (8) Wash the DNA precipitate twice with 75% ethanol, dry it, and then add TE buffer to dissolve the DNA to obtain the extracted genomic DNA sample.

[0022] Furthermore, in the S100, the DNA concentration in the multi-source heterogeneous data collection is ≥20 ng / μL, and the OD260 / 280 is between 1.8-2.0.

[0023] Furthermore, the upstream primer sequence of the monkey Campylobacter jejuni CJ is shown in SEQ ID No. 1, which is 5'-CTATAACAACTGCACCTACTAAT-3', the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-ATGAAATTTTTGCCAGTGGTG-3', and the probe sequence is shown in SEQ ID No. 3, which is 5'-FAM-CTTAATAGCCGTCACCCCAC-BHQ1-3'; the upstream primer sequence of the monkey enterocolitica YE is shown in SEQ ID No. 4, which is 5'-GCATTAACGAATATGTTAGC-3', the downstream primer sequence is shown in SEQ ID No. 5, which is 5'-ATCGAGTTTGGAGTATTCAT-3', and the probe sequence is shown in SEQ ID No. 6, which is 5'-VIC-CCGCTTCCAAATTTTGTCAT-BHQ1-3'.

[0024] Furthermore, in the preparation of the dual digital PCR reaction system in S300, the 2× digital PCR premix solution comprises a hot start Taq enzyme, dNTPs, MgCl2 and a stabilizer, wherein Mg 2+ The final concentration is 3-5mM.

[0025] Furthermore, in the step S400 , the pressure parameter of the droplet generator in the droplet generation and distribution is set to 35-40 psi, the generation time is 2-3 minutes, and the number of droplets per well is ≥15,000.

[0026] Furthermore, in the S600, fluorescence signal detection and data analysis, a dual-channel scanner is used for fluorescence signal detection, with an FAM excitation wavelength of 488 nm and an emission wavelength of 525 nm for detecting CJ; and a VIC excitation wavelength of 532 nm and an emission wavelength of 575 nm for detecting YE.

[0027] Furthermore, in the step S600, the Poisson distribution formula is used to calculate the copy number concentration in the fluorescence signal detection and data analysis. The Poisson distribution formula is: Where P is the number of positive droplets, N is the total number of droplets, and V is the reaction volume.

[0028] Furthermore, in the step S600, for fluorescence signal detection and data analysis, if the total number of droplets is less than 10,000, the droplet breakage rate is greater than 10%, or the overlap of the positive and negative droplet populations is greater than 20%, the droplets must be re-prepared. 11. Furthermore, in the step S600, for fluorescence signal detection and data analysis, positive controls, negative controls, and internal reference controls are set for each test. The positive control contains plasmid DNA of known concentration with a copy number deviation of ≤±20%. The negative control is nuclease-free water. The internal reference control is the monkey β-actin gene, whose upstream primer sequence is shown in SEQ ID No. 7, 5'-CCTGGCACCCAGCACAAT-3', the downstream primer sequence is shown in SEQ ID No. 8, 5'-GCCGATCCACACGGAGTACT-3', and the probe sequence is shown in SEQ ID No. 9, 5'-Cy5-TGCTGCTGACGTCATCGTCC-BHQ2-3'. The positive rate must be ≥95%, otherwise the test must be repeated.

[0029] Compared with the existing technology, the dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica has the following beneficial effects:

[0030] 1. The present invention achieves highly sensitive and specific detection of two pathogens through a carefully designed primer and probe combination. This method utilizes the conserved regions of the gyrA gene of monkey Campylobacter jejuni CJ and the POL gene of monkey Yersinia enterocolitica YE as detection targets, amplifying specific fragments of 249 bp and 182 bp, respectively, effectively avoiding interference from nonspecific amplification. Dual digital PCR technology not only enables simultaneous detection of two pathogens in a single reaction system, but also accurately calculates the target gene copy number concentration using the Poisson distribution formula, thereby improving the accuracy and reliability of detection. Furthermore, fluorescence signal detection utilizes a dual-channel scanner, utilizing the FAM and VIC channels to detect CJ and YE, respectively, further enhancing the specificity and sensitivity of detection. This dual detection capability of high sensitivity and specificity provides strong support for rapid clinical diagnosis and scientific research, facilitating the timely implementation of prevention and control measures to reduce the spread and harm of pathogens.

[0031] 2. The present invention extracts genomic DNA from monkey feces and intestinal tissue samples through multi-source heterogeneous data collection, and undergoes strict concentration and purity testing to ensure sample quality. During the preparation of the dual digital PCR reaction system, the dosage of each component is accurate, and subsequent experiments can be carried out after mixing and centrifugation. The droplet generation and distribution, and digital PCR amplification program steps ensure the stability and repeatability of the experiment. Fluorescence signal detection and data analysis use advanced dual-channel scanners and Poisson distribution formulas, which can quickly and accurately obtain test results. In addition, this method also sets positive controls, negative controls, and internal reference controls to further ensure the reliability of the test results.

[0032] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0034] Figure 1 A flow chart of a dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica;

[0035] Figure 2 Establish amplification profiles for dual droplet digital PCRCJ;

[0036] Figure 3 Establish amplification profiles for dual droplet digital PCR;

[0037] Figure 4 Optimize the amplification profile for duplex droplet digital PCR annealing temperature (CJ);

[0038] Figure 5 Amplification profiles for optimized annealing temperature for duplex droplet digital PCR (YE). DETAILED DESCRIPTION

[0039] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0040] Example 1:

[0041] Materials and methods: Figure 1 shown

[0042] Samples and reagents: Pathogens and DNA samples: 8 virus strains stored in the laboratory, including Streptococcus pneumoniae, Pasteurella pneumophila H-type, Pasteurella pneumophila J-type, Corynebacterium rapae, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa, were used to extract their genomic DNA for specific experiments.

[0043] Primers and probes:

[0044] CJ primer probe: The upstream primer sequence is shown in SEQ ID No. 1, which is 5'-CTATAACAACTGCACCTACTAAT-3'; the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-ATGAAATTTTTGCCAGTGGTG-3'; the probe sequence is shown in SEQ ID No. 3, which is 5'-FAM-CTTAATAGCCGTCACCCCAC-BHQ1-3'; and the amplified fragment is 249 bp.

[0045] YE primer probe: The upstream primer sequence is shown in SEQ ID No. 4, which is 5'-GCATTAACGAATATGTTAGC-3'; the downstream primer sequence is shown in SEQ ID No. 5, which is 5'-ATCGAGTTTGGAGTATTCAT-3'; the probe sequence is shown in SEQ ID No. 6, which is 5'-VIC-CCGCTTCCAAATTTTGTCAT-BHQ1-3'; the amplified fragment is 182 bp; and the storage concentration of both is 10 μM.

[0046] Positive quality control: construct recombinant plasmids using the PCR amplified target fragments of CJ and YE as positive controls.

[0047] Establishment of droplet digital PCR method:

[0048] DNA extraction: Genomic DNA from monkey feces and intestinal tissue samples was extracted using the phenol-chloroform method. The specific steps are as follows:

[0049] The sample was ground into powder with liquid nitrogen, and lysis buffer containing SDS and proteinase K was added, and incubated in a 56°C water bath for 1-2 hours.

[0050] An equal volume of phenol-chloroform-isoamyl alcohol mixture was added, mixed by inversion for 10-15 minutes, centrifuged at 12000 rpm for 10 minutes, and the upper aqueous phase was aspirated.

[0051] Repeat the extraction 1-2 times until there is no protein precipitation at the interface. Add 0.1 times the volume of 3M sodium acetate and 2 times the volume of anhydrous ethanol to the supernatant and precipitate at -20℃ for 1-2 hours.

[0052] Centrifuge at 12000 rpm for 15 minutes, wash the DNA precipitate twice with 75% ethanol, and dissolve it in TE buffer. The DNA concentration should be ≥20 ng / μL and the OD260 / 280 should be between 1.8 and 2.0.

[0053] Reaction system (20 μL): 2× digital PCR premix 10 μL (containing hot start Taq enzyme, dNTPs, MgCl2, Mg 2+ Final concentration 3-5 mM).

[0054] 2 μL of DNA template.

[0055] 0.6 μL each of CJ upstream and downstream primers (final concentration 600 nM), 0.6 μL each of YE upstream and downstream primers (final concentration 600 nM).

[0056] 0.2 μL of CJ probe (final concentration 200 nM), 0.2 μL of YE probe (final concentration 200 nM).

[0057] Add nuclease-free pure water to 20 μL, mix well and centrifuge.

[0058] Droplet generation and dispensing:

[0059] The reaction solution and droplet generation oil were added to the droplet generation chip in a ratio of 1:2. The reaction system was added to the sample well and 70 μL of digital PCR oil was added to the oil phase well.

[0060] The pressure parameter of the droplet generator was set to 35-40 psi, the generation time was 2-3 minutes, and the number of droplets per well was ≥15,000.

[0061] Amplification procedure:

[0062] Pre-denaturation at 95°C for 15 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, 40 cycles; final denaturation at 98°C for 10 minutes, hold at 20°C for 2 minutes, temperature ramp rate 2.0°C / s. After the reaction, the chip was placed in a scanner, the instrument automatically collected fluorescence signals, and the software GeneCountAnalysisSystem was used to read the DNA copy number. The results are shown in Table 1. Figure 2 and Figure 3 .

[0063]

[0064] Table 1: Data statistics of droplet digital PCR

[0065] Fluorescence signal detection:

[0066] Dual-channel scanner detection, FAM excitation wavelength 488nm, emission wavelength 525nm (detection CJ), VIC excitation wavelength 532nm, emission wavelength 575nm (detection YE).

[0067] The copy number concentration was calculated using the Poisson distribution formula, which is: Where P is the number of positive droplets, N is the total number of droplets, and V is the reaction volume.

[0068] Judgment criteria: ≥3 positive droplets in the CJ or YE channel and a concentration ≥3.0 copies / μL are considered positive, otherwise negative. If the total number of droplets is <10,000, the breakage rate is >10%, or the overlap of positive and negative droplet groups is >20%, the droplets need to be re-prepared.

[0069] Method optimization and validation:

[0070] Annealing temperature optimization: The tested annealing temperatures were 51.8°C, 53.2°C, 54.6°C, 56.0°C, 57.4°C, 58.8°C, 60.2°C, and 61.7°C, the primer concentration was 20 μM, and the probe concentration was 10 μM. As shown in Table 2, Figure 4 and Figure 5 The annealing temperature shown had no significant effect on the number of oil droplets (total oil droplets, positive oil droplets), and the amplification effect was best when the annealing temperature was around 55°C.

[0071]

[0072] Table 2: Statistics of droplet digital PCR annealing temperature optimization data

[0073] Reaction system optimization: 12 different primer-probe concentration combinations (systems 1-12) were tested.

[0074] The reaction system was 25 μL.

[0075] System 1: Contains 10.0 μL of digital PCR premix, 1.0 μL each of CJ upstream and downstream primers (20 μM), 0.2 μL of CJ probe (20 μM), 1.0 μL each of YE upstream and downstream primers (20 μM), 0.2 μL of YE probe (20 μM), 3.6 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0076] System 2: Contains 10.0 μL of digital PCR premix, 0.8 μL each of CJ upstream and downstream primers (20 μM), 0.2 μL of CJ probe (20 μM), 0.8 μL each of YE upstream and downstream primers (20 μM), 0.2 μL of YE probe (20 μM), 4.4 μL of RNase-free ddH2O, and 4 μL of template DNA.

[0077] System 3: Contains 10.0 μL of digital PCR premix, 0.6 μL each of CJ upstream and downstream primers (20 μM), 0.2 μL of CJ probe (20 μM), 0.6 μL each of YE upstream and downstream primers (20 μM), 0.2 μL of YE probe (20 μM), 5.2 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0078] System 4: Contains 10.0 μL of digital PCR premix, 0.4 μL each of CJ upstream and downstream primers (20 μM), 0.2 μL of CJ probe (20 μM), 0.4 μL each of YE upstream and downstream primers (20 μM), 0.2 μL of YE probe (20 μM), 6.0 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0079] System 5: Contains 10.0 μL of digital PCR premix, 1.0 μL each of CJ upstream and downstream primers (20 μM), 0.5 μL of CJ probe (20 μM), 1.0 μL each of YE upstream and downstream primers (20 μM), 0.5 μL of YE probe (20 μM), 3.0 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0080] System 6: Contains 10.0 μL of digital PCR premix, 0.8 μL each of CJ upstream and downstream primers (20 μM), 0.5 μL of CJ probe (20 μM), 0.8 μL each of YE upstream and downstream primers (20 μM), 0.5 μL of YE probe (20 μM), 3.8 μL of RNase-free ddH2O, and 5 μL of template DNA.

[0081] System 7: Contains 10.0 μL of digital PCR premix, 0.6 μL each of CJ upstream and downstream primers (20 μM), 0.5 μL of CJ probe (20 μM), 0.6 μL each of YE upstream and downstream primers (20 μM), 0.5 μL of YE probe (20 μM), 4.6 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0082] System 8: Contains 10.0 μL of digital PCR premix, 0.4 μL each of CJ upstream and downstream primers (20 μM), 0.5 μL of CJ probe (20 μM), 0.4 μL each of YE upstream and downstream primers (20 μM), 0.5 μL of YE probe (20 μM), 5.4 μL of RNase-free ddH2O, and 5 μL of template DNA.

[0083] System 9: Contains 10.0 μL of digital PCR premix, 1.0 μL each of CJ upstream and downstream primers (20 μM), 0.8 μL of CJ probe (20 μM), 1.0 μL each of YE upstream and downstream primers (20 μM), 0.8 μL of YE probe (20 μM), 2.4 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0084] System 10: Contains 10.0 μL of digital PCR premix, 0.8 μL each of CJ upstream and downstream primers (20 μM), 0.8 μL of CJ probe (20 μM), 0.8 μL each of YE upstream and downstream primers (20 μM), 0.8 μL of YE probe (20 μM), 3.2 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0085] System 11: Contains 10.0 μL of digital PCR premix, 0.6 μL each of CJ upstream and downstream primers (20 μM), 0.8 μL of CJ probe (20 μM), 0.6 μL each of YE upstream and downstream primers (20 μM), 0.8 μL of YE probe (20 μM), 4.0 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0086] System 12: Contains 10.0 μL of digital PCR premix, 0.4 μL each of CJ upstream and downstream primers (20 μM), 0.8 μL of CJ probe (20 μM), 0.4 μL each of YE upstream and downstream primers (20 μM), 0.8 μL of YE probe (20 μM), 4.8 μL of RNase-free ddH2O, and 2 μL of template DNA.

[0087] Oil droplets were generated and amplified according to the groups described above. The amplification program was as follows: 95°C for 15 minutes; 94°C for 30 seconds, annealing at 55°C for 1 minute, for a total of 40 cycles; 98°C for 10 minutes; and a hold at 20°C for 2 minutes. The reaction temperature was ramped at a rate of 2.0°C / s. After the reaction, the chip was placed in a scanner, and the instrument automatically acquired the fluorescence signal. DNA copy number was determined using the GeneCount Analysis System software. Groups 1 through 4 were able to distinguish between positive and negative results. In Group 3, the CV values ​​for both the FAM and VIC channels were <15%. Group 3 was selected as the primer and probe concentration for duplex ddPCR of CJ and YE, as shown in Table 3.

[0088]

[0089]

[0090]

[0091] Table 3: Statistics of droplet digital PCR system optimization data

[0092] Sensitivity and minimum detection limit: Combine the CJ and YE positive quality control products and dilute them 10-fold. The theoretical concentrations are 185,000, 18,500, 1850, 185, 18.5, 1.85, and 0.185 copies / μL. Repeat the test three times.

[0093] The results showed that the minimum detection limit of CJ was 3.2 copies / μL, and that of YE was 3.1 copies / μL. The relative standard deviations (RSDs) of the detection results for each gradient were all less than 10%.

[0094] Specificity detection: Using the DNA of five pathogens, including rhesus cytomegalovirus, simian varicella virus, measles virus, and simian retrovirus D, as templates, the results showed that the positive quality control (recombinant plasmid) was positive, and the nucleic acid tests of other pathogens were not positive. The specific results are shown in Table 4.

[0095]

[0096] Table 4: Droplet digital PCR specificity data statistics

[0097] Minimum detection limit test results: The positive quality control product (recombinant plasmid) was diluted 10-fold or 2-fold, and the theoretical concentrations of each dilution factor were 30, 15, and 7.5 copies / μL, respectively. The optimized ddPCR method was used for detection. The minimum detection limit of the CJ test was 3.2 copies / μL, and the minimum detection limit of the YE test was 3.1 copies / μL. The test results are shown in Table 5.

[0098]

[0099]

[0100] Table 5: Duplex ddPCR standard curve and minimum detection limit raw data

[0101] Repeatability verification: The positive quality control product was diluted 10-fold to 3,000,000, 300,000, 30,000, 3,000, and 300 copies / μL, with each group repeated three times. The optimized ddPCR was used for detection, and each dilution concentration was repeated three times. The test results are shown in Table 6.

[0102]

[0103] Table 6: Raw data of ddPCR repeatability test

[0104] Summary of the testing process:

[0105] Sample DNA was extracted and quality tested (concentration ≥ 20 ng / μL, OD260 / 280 1.8-2.0).

[0106] The reaction solution was prepared according to the optimized 20 μL system to generate microdroplets (≥15,000 per well).

[0107] The amplification program was performed as follows: 95°C for 15 min → (94°C for 30 s, 55°C for 1 min) × 40 cycles → 98°C for 10 min → 20°C for 2 min.

[0108] The fluorescence signal was detected by dual-channel scanning, and the copy number was calculated using Poisson distribution. A positive result was determined when the number of positive droplets was ≥3 and the concentration was ≥3.0 copies / μL.

[0109] In summary, the dual digital PCR detection method provided by the present invention uses the phenol-chloroform method to extract sample DNA. Specific primers and probes are designed based on the gyrA gene of CJ and the POL gene of YE. The reaction system and amplification conditions are optimized to establish an efficient detection system. In a 20 μL system, 600 nM primers and 200 nM probes are annealed at 55°C to generate ≥15,000 droplets. The copy number is calculated using a Poisson distribution, with a positive criterion of ≥3 positive droplets and a concentration ≥3.0 copies / μL. The minimum detection limits for CJ and YE have been verified to be 3.2 and 3.1 copies / μL, respectively. There is no cross-reactivity with other pathogens, and the copy number deviation of repeatable detection is ≤±20%. This method overcomes the time-consuming nature of traditional culture methods and the low sensitivity of conventional PCR, achieving absolute quantification of monkey CJ and YE, providing an efficient solution for primate pathogen detection.

[0110] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica, characterized in that: The specific steps of this detection method are: S100, multi-source heterogeneous data acquisition: monkey feces and intestinal tissue samples were collected, genomic DNA was extracted using the phenol-chloroform method, DNA concentration and purity were tested, and after reaching the standard, it was stored at -20°C for future use; S200, primer and probe design and synthesis: Primers and probes were designed based on the conserved regions of the gyrA gene of monkey Campylobacter jejuni CJ and the POL gene of monkey Yersinia enterocolitica YE. The CJ primer-probe set amplified a 249 bp fragment, and the YE primer-probe set amplified a 182 bp fragment. The primers and probes were prepared into a 10 μM storage solution and stored at -20°C. S300, preparation of duplex digital PCR reaction system: prepare 20 μL of system on ice, containing 10 μL 2× digital PCR premix, 2 μL DNA template, 0.6 μL each of CJ and YE upstream and downstream primers, and 0.2 μL each of CJ and YE probes. Make up with nuclease-free pure water, mix well, and centrifuge; S400, droplet generation and distribution: The reaction solution and droplet generation oil are added to the droplet generation chip at a ratio of 1:

2. The reaction system is added to the sample well, 70 μL of digital PCR oil is added to the oil phase well, and the chip is placed in the droplet generator to generate water-in-oil droplets. S500, digital PCR amplification program: transfer the droplet to a PCR tube, pre-denaturation at 95°C for 15 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, 40 cycles, final denaturation at 98°C for 10 minutes, and then hold at 20°C for 2 minutes, with a temperature ramp rate of 2.0°C / s; S600, fluorescence signal detection and data analysis: A fluorescence scanner is used to detect FAM and VIC fluorescence signals, the number of positive droplets is counted, and the target gene copy number concentration is calculated based on the Poisson distribution. A positive result is determined if there are ≥3 positive droplets in the CJ or YE channel and the concentration is ≥3.0 copies / μL; otherwise, it is considered negative.

2. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: The specific steps of extracting sample genomic DNA using the phenol-chloroform method are: (1) Place the collected feces and intestinal tissue samples in a mortar and add liquid nitrogen to grind them thoroughly until they become powder; (2) Transfer the ground powder to a centrifuge tube, add lysis buffer containing SDS and proteinase K, mix gently, and incubate in a 56°C water bath for 1-2 hours to lyse the cells and release genomic DNA; (3) Add an equal volume of phenol-chloroform-isoamyl alcohol mixture to the centrifuge tube and mix by inversion for 10-15 minutes to denature the protein and separate the nucleic acid; (4) Centrifuge at 12000 rpm for 10 minutes at room temperature. After the solution is separated, transfer the upper aqueous phase to a new centrifuge tube. (5) Repeat steps (3)-(4) 1-2 times until there is no obvious protein precipitation at the interface after centrifugation; (6) Add 0.1 volumes of 3 M sodium acetate and 2 volumes of anhydrous ethanol to the supernatant, mix thoroughly by inversion, and precipitate in a -20°C refrigerator for 1-2 hours; (7) Centrifuge at 12000 rpm for 15 minutes, discard the supernatant, and retain the DNA pellet at the bottom of the tube; (8) Wash the DNA precipitate twice with 75% ethanol, dry it, and then add TE buffer to dissolve the DNA to obtain the extracted genomic DNA sample.

3. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: In the S100, the DNA concentration in multi-source heterogeneous data collection is ≥20 ng / μL, and the OD260 / 280 is between 1.8 and 2.

0.

4. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: The upstream primer sequence of the monkey Campylobacter jejuni CJ is shown in SEQ ID No. 1, which is 5'-CTATAACAACTGCACCTACTAAT-3', the downstream primer sequence is shown in SEQ ID No. 2, which is 5'-ATGAAATTTTTGCCAGTGGTG-3', and the probe sequence is shown in SEQ ID No. 3, which is 5'-FAM-CTTAATAGCCGTCACCCCAC-BHQ1-3'; the upstream primer sequence of the monkey enterocolitica YE is shown in SEQ ID No. 4, which is 5'-GCATTAACGAATATGTTAGC-3', the downstream primer sequence is shown in SEQ ID No. 5, which is 5'-ATCGAGTTTGGAGTATTCAT-3', and the probe sequence is shown in SEQ ID No. 6, which is 5'-VIC-CCGCTTCCAAATTTTGTCAT-BHQ1-3'.

5. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 4, characterized in that: In the S300, the dual digital PCR reaction system is prepared with a 2× digital PCR premix comprising a hot start Taq enzyme, dNTPs, MgCl2 and a stabilizer, wherein Mg 2+ The final concentration is 3-5mM.

6. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 4, characterized in that: In the step S400, the pressure parameter of the droplet generator in the droplet generation and distribution is set to 35-40 psi, the generation time is 2-3 minutes, and the number of droplets per well is ≥15,000.

7. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: In the S600, fluorescence signal detection and data analysis, a dual-channel scanner is used for fluorescence signal detection, with an FAM excitation wavelength of 488 nm and an emission wavelength of 525 nm for detecting CJ; and a VIC excitation wavelength of 532 nm and an emission wavelength of 575 nm for detecting YE.

8. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: In the step S600, the Poisson distribution formula is used to calculate the copy number concentration in the fluorescence signal detection and data analysis. The Poisson distribution formula is: Where P is the number of positive droplets, N is the total number of droplets, and V is the reaction volume.

9. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: In the step S600, fluorescence signal detection and data analysis, if the total number of droplets is less than 10,000, the droplet breakage rate is greater than 10%, or the overlap of the positive and negative droplet groups is greater than 20%, the droplets need to be re-prepared.

10. The dual digital PCR detection method for monkey Campylobacter jejuni and monkey Yersinia enterocolitica according to claim 1, characterized in that: In the S600, in the fluorescence signal detection and data analysis, a positive control, a negative control, and an internal reference control are set for each detection. The positive control contains plasmid DNA of known concentration with a copy number deviation of ≤±20%. The negative control is nuclease-free water. The internal reference control is monkey β-actin gene, whose upstream primer sequence is shown in SEQ ID No. 7, which is 5'-CCTGGCACCCAGCACAAT-3', the downstream primer sequence is shown in SEQ ID No. 8, which is 5'-GCCGATCCACACGGAGTACT-3', and the probe sequence is shown in SEQ ID No. 9, which is 5'-Cy5-TGCTGCTGACGTCATCGTCC-BHQ2-3'. The positive rate must be ≥95%, otherwise the experiment is repeated.

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