Quantitative detection method and primer group for tiny difference of copy number of specific DNA (Deoxyribose Nucleic Acid) sequence

Abstract

The invention relates to a quantitative detection method for tiny difference of specific DNA sequence copy numbers and a primer group. The method comprises the following steps: 1) extracting sample DNA as a detection template; 2) preparing a digital PCR reaction premixed solution; (3) preparing micro-droplets; 4) performing PCR reaction; 5) detecting the amplification product by using a biochip analyzer, and judging the copy number of the target gene amplified by the primer composition; the method meets the requirement of six-color 10-order signal reading, hundreds of times of signal interpretation can be realized by improving amplification specificity, the quantitative detection requirement on tiny difference of specific DNA sequence copy numbers is met, the method is not influenced by complex components in a sample, and the accuracy of a detection result and the detection efficiency can be remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for quantitatively detecting slight differences in the copy number of a specific DNA sequence. Background Art

[0002] dPCR technology, also known as third-generation PCR technology, is a new method for highly sensitive nucleic acid detection and absolute quantification. Compared to traditional PCR, dPCR divides a reaction system of tens of microliters into tens of thousands of tiny independent reaction systems. The nucleic acid template is fully diluted during this separation process. Ideally, each droplet contains one molecule of nucleic acid template. After amplification is completed, the fluorescent signals of all droplets are identified and counted, and the number of negative and positive reactions is calculated. The concentration of the target molecule is calculated using the Poisson distribution principle. This operation of separating the reaction system improves the specificity of amplification and is not affected by the efficiency of PCR amplification. It has higher sensitivity and accuracy, can realize single-molecule detection and small difference detection, and does not rely on standard curve quantification, thereby achieving absolute quantification of target molecules.

[0003] However, dPCR technology still has limitations in detecting targets. Although dPCR can perform quantitative analysis, when simultaneous quantitative detection of multiple sites is required, the location, number, and distribution of the detection targets will also affect the accuracy of the quantification. The more detection sites, the higher the detection accuracy. Therefore, solving the detection throughput problem of dPCR is the key to expanding the application limits of dPCR. However, due to common problems of PCR-based technologies such as interference between different target amplifications caused by non-specific amplification and insufficient resolution of negative and positive signals, most current platforms use fluorescent color multiplexing only to allow each fluorescent channel to identify one target. Expanding the number of detection targets by adding multiple fluorescent channels can only detect 6-8 targets at most simultaneously, and this will increase the cost of the detection system. When trying to increase the detection throughput, because dPCR has limitations in distinguishing by signal intensity, it is usually mainly used to distinguish between positive and negative. By design, it can generally only distinguish between strong positive, medium positive, weak positive, and negative. If multiple fluorescent channels are combined with multiple signal intensity distinctions, most dPCR detection products can only achieve simultaneous detection of no more than 20 targets, which is far from meeting the detection needs in practice.

[0004] Furthermore, in the analysis of plant and animal-derived ingredients in food, digital PCR technology can perform absolute quantitative detection of animal-derived ingredients, independent of standard curves, unaffected by amplification efficiency, and with minimal PCR inhibition. However, quantitative analysis strategies based on digital PCR are still affected by factors such as result presentation, target gene selection, DNA extraction efficiency, DNA degradation, and species genome size ("Research Progress on Quantitative Detection of Animal-Derived Components in Meat Products Based on Real-Time Fluorescence Polymerase Chain Reaction Technology," Ma Bingcun et al., Meat Research, 2021). Animal mitochondrial DNA has high interspecies diversity and low intraspecies variation. Although it can be used for source analysis, its high copy number in cells makes it difficult to achieve "high-throughput" detection with existing technologies.

[0005] In the detection of foodborne microorganisms, the traditional method involves culturing microorganisms to a certain number before testing, which involves steps such as proliferation, isolation, and identification, and is time-consuming and labor-intensive. While qPCR, which designs primers specific to the target gene sequence of pathogens and performs PCT amplification, has achieved significant time savings, this method suffers from significant deviations in final test results when the target molecule is low and the detection matrix is ​​complex. The accuracy of the results depends on the construction of the standard curve and the amplification efficiency of the primers, limiting its application in pathogen detection ("Research Progress in the Application of Digital PCR in Biological Detection," Huang Jin et al., Life Sciences [J], 2021). dPCR technology, leveraging its ability to resist inhibition, can be a useful tool for high-throughput screening of microorganisms to assess food quality and safety. However, for the detection of multiple pathogens, the throughput limitations of existing dPCR detection technology make it difficult to meet the requirements for detecting multiple bacterial species simultaneously.

[0006] Therefore, improving the high-throughput technology of simultaneous detection of multiple targets by dPCR (the number of targets in a single detection reaches hundreds or thousands) has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0007] In response to the above problems, this application establishes a high-throughput multi-target PCR detection method based on digital PCR technology. On the basis of traditional digital PCR, it solves the throughput limitation of its detection technology, realizes the detection of multiple targets at one time, and performs quantitative analysis of small variations in the copy number of multiple DNA fragments at one time, filling the technical needs of various molecular detection scenarios.

[0008] This application is achieved through the following technical solutions:

[0009] First, this application provides a method for quantitatively detecting slight differences in the copy number of a specific DNA sequence, the specific steps of which are as follows:

[0010] 2) Extract sample DNA as a template for subsequent PCR testing;

[0011] 2) Prepare digital PCR reaction system,

[0012] The PCR reaction system includes a DNA template, a primer-probe combination targeting the target sequence, a reaction substrate, and a polymerase.

[0013] PCR primers were designed for the target sites, and a digital PCR reaction premix was prepared: 10 μL of 2× digital PCR premix, 1.0 μL of enzyme (1500 U / ml), 5 μL of primer-probe mixture (final concentration 10 μM), 10 ng of detection template, and the mixture was made up to 20 μL with nuclease-free water.

[0014] 3) Preparation of microdroplets

[0015] Using a fully automated sample processing system, add 20 μL of the digital PCR reaction premix prepared in step 2) to the sample well to generate droplets; then transfer the droplets to the PCR reaction plate;

[0016] 6) PCR reaction

[0017] Placing the PCR reaction plate described in step 3) in a nucleic acid amplifier to perform a PCR reaction to obtain an amplified product;

[0018] The reaction procedure was as follows: step 1: 25°C, reaction for 10 min, 1 cycle; step 2: 95°C, reaction for 10 min, 1 cycle; step 3: 95°C for 10 s, 62°C for 50 s, 55 cycles; step 4: 98°C for 10 min, 1 cycle; step 5: 16°C for 1 cycle;

[0019] 7) Detecting the amplified product using a biochip analyzer to determine the copy number of the target gene amplified by the primer combination.

[0020] In addition, the present application also provides a PCR primer set for analyzing animal-derived components in food (preferably muscle tissue), the nucleotide sequences of which are shown in SEQ ID NO.1-SEQ IN NO.36; in actual application, the 5' ends of primers SEQ IN NO.3 and SEQ IN NO.6 are both connected to a FAM fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.9 and SEQ IN NO.12 are both connected to a VIC fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.15 and SEQ IN NO.18 are both connected to a ROX fluorescent group, and the 3' ends are both connected to a BHQ2 fluorescent group; the 5' ends of primers SEQ IN NO.21 and SEQ IN NO.24 are both connected to a CY5 fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.27 and SEQ IN NO.30 are both connected to a CY5.5 fluorescent group, and the 3' ends are both connected to a BHQ3 fluorescent group; The 5' ends of SEQIN NO.33 and SEQIN NO.36 were both connected to the CY7 fluorescent group, and the 3' ends were both connected to the BHQ3 fluorescent group.

[0021] A PCR primer set for detecting foodborne pathogens, the nucleotide sequences of which are shown in SEQ ID NO.37-SEQ ID NO.90. In practical applications, the 5' ends of primers SEQ IN NO.39, SEQ IN NO.42, and SEQ IN NO.45 designed for Listeria monocytogenes are all connected to FAM fluorescent groups, and the 3' ends are all connected to BHQ1 fluorescent groups; the 5' ends of primers SEQ IN NO.48, SEQ IN NO.51, and SEQ IN NO.54 designed for Salmonella are all connected to CY5 fluorescent groups, and the 3' ends are all connected to BHQ2 fluorescent groups; the 5' ends of primers SEQ IN NO.57, SEQ IN NO.60, and SEQ IN NO.63 designed for Vibrio parahaemolyticus are all connected to VIC fluorescent groups, and the 3' ends are all connected to BHQ1 fluorescent groups; the 5' ends of primers SEQ IN NO.66, SEQ IN NO.69, and SEQ IN NO.72 designed for Staphylococcus aureus are all connected to ROX fluorescent groups, and the 3' ends are all connected to BHQ2 fluorescent groups; the 5' ends of primers SEQ IN NO.75, SEQ IN NO.78, and SEQ IN NO.79 designed for Shiga toxin-producing Escherichia coli are all connected to CY5 fluorescent groups, and the 3' ends are all connected to BHQ2 fluorescent groups. The 5' end of primer INNO.81 is connected to the CY7 fluorescent group, and the 3' end is connected to the BHQ3 fluorescent group; the 5' end of primers SEQ IN NO.84, SEQ IN NO.87, and SEQ IN NO.90 designed for Bacillus cereus are connected to the CY5.5 fluorescent group, and the 3' end is connected to the BHQ3 fluorescent group.

[0022] A PCR primer set for detecting the copy number of male Y chromosome, the nucleotide sequences of which are shown in SEQ ID NO.91-SEQ ID NO.269. In practical applications, the primers for Y chromosome detection are SEQ IN NO.93, SEQ IN NO.96, SEQ IN NO.99, SEQ IN NO.102, SEQ IN NO.105, SEQ IN NO.108, SEQ IN NO.110, SEQ IN NO.113, SEQ IN NO.116, SEQ IN NO.119, SEQ IN NO.122, SEQ IN NO.125, SEQ IN NO.128, SEQ IN NO.131, SEQ IN NO.134, SEQ IN NO.137, SEQ IN NO.140, SEQ IN NO.143, SEQ IN NO.146, SEQ IN NO.149, SEQ IN NO.152, SEQ IN NO.155, SEQ IN NO.158, SEQ IN NO.161, SEQ IN NO.164、SEQ IN NO.167、SEQ IN NO.170、SEQ IN NO.173、SEQ IN NO.176、SEQ The 5' end of INNO.179 is connected to the VIC fluorescent group, and the 3' end is connected to the BHQ1 fluorescent group. Primers for chromosome 1 detection: SEQ IN NO.182, SEQ IN NO.185, SEQ IN NO.188, SEQ IN NO.191, SEQ IN NO.194, SEQ IN NO.197, SEQ IN NO.200, SEQ IN NO.203, SEQ IN NO.206, SEQ IN NO.209, SEQ IN NO.212, SEQ IN NO.215, SEQ IN NO.218, SEQ IN NO.221, SEQ IN NO.224, SEQ IN NO.227, SEQ IN NO.230, SEQ IN NO.233, SEQ IN NO.236, SEQ IN NO.239, SEQ IN NO.242, SEQ IN NO.245, SEQ IN NO.248, SEQ IN NO.251, SEQ IN NO.254、SEQ IN NO.257、SEQ IN NO.260、SEQ INNO.263、SEQ IN NO.266、SEQ IN The 5' end of NO.269 is connected to the FAM fluorescent group, and the 3' end is connected to the BHQ1 fluorescent group.

[0023] This application provides a detection method and primer set based on high-throughput dPCR technology for quantitative analysis of small differences in the copy number of specific DNA fragments. The selected high-throughput dPCR technology is based on a method that meets the requirements of 6-color 10-order signal reading. By improving amplification specificity, it can achieve hundreds of multiple signal interpretations, meeting the requirements for quantitative detection of small differences in the copy number of specific DNA sequences. Compared with existing technologies, the method of this application has the following advantages:

[0024] 1) The detection method established in this application adopts high-throughput PCR detection technology. Based on the high sensitivity (can detect rare mutations with a mutation frequency of less than 0.01%) and absolute quantification (can identify copy number changes with small differences) of dPCR technology, it can achieve simultaneous detection of hundreds of detection sites. At the same time, through multi-target detection, the accuracy of the test results can be significantly improved.

[0025] 2) Compared with the existing fluorescence quantitative PCR detection method, the method for detecting animal-derived ingredients such as cattle, sheep, and pigs in meat products established in this application does not require the establishment of a standard curve for each animal-derived ingredient, and multiple test samples can be tested in a "one tube", which is simple to operate and has higher detection efficiency.

[0026] 3) The method developed in this application for simultaneous quantitative detection of multiple pathogenic bacterial DNA in food is simpler to use than currently used traditional culture methods. It can detect trace amounts of target DNA fragments without the need for culture, resulting in faster detection. Compared with qPCR, it offers higher sensitivity and can simultaneously detect multiple bacterial species, unaffected by complex sample components, resulting in higher accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the correlation between the number of primer pairs and the Y chromosome ratio measurement accuracy.

[0028] Figure 2 This is the result of chromosome Y detection in Example. DETAILED DESCRIPTION

[0029] Example 1 Analysis of animal-derived ingredients in food

[0030] Mitochondrial DNA has high interspecies diversity and low intraspecies variation, and its copy number is high in cells. Therefore, it can serve as a detection target for the quantitative analysis of animal-derived components in meat products. This example establishes a method for detecting animal-derived components in meat products from pigs, sheep, cattle, rabbits, and mice. By quantitatively analyzing different animal-derived components in the test sample, the presence of other animal-derived components in the meat product can be assessed. In this example, the same fluorescent marker is selected for detection of the same species to facilitate quantitative analysis.

[0031] The primer and probe design scheme is shown in Table 1:

[0032] Table 1 Primers

[0033]

[0034]

[0035] In Table 1, FAM, BHQ1, VIC, ROX, BHQ2, CY5, CY5.5, BHQ3, and CY7 are all conventional fluorescent groups.

[0036] The experimental steps are as follows:

[0037] S1. DNA extraction from samples: Muscle tissue was collected at multiple points and homogenized. DNA was extracted from the homogenized samples. For specific methods, refer to the instructions of the Nucleic Acid Extraction Kit (Centrifugal Column Method) (Puji Biotechnology).

[0038] 1) Sample Processing: After removing the sample, equilibrate it to room temperature and add 200 μL of sample release agent (Nanjing Puji Biomedical, see the product manual for the addition ratio). 1 g of sample was added to a grinding medium tube, followed by 300 μL of PBS. Using a JXFSTPRP-4DL grinder (Shanghai Jingxin Industrial Development Co., Ltd.), the sample was processed and spun at 18 m / s for 10 cycles of 30 s, with 10 s intervals. After grinding, the pathogen lysis tube was removed and centrifuged at 6000 × g for 5 s. 400 μL of the supernatant was transferred to a clean 1.5 mL EP tube for subsequent experiments.

[0039] 2) Add 400 μL of whole blood sample to a 1.5 mL centrifuge tube, add 40 μL of proteinase K (type C), vortex mix for 10 seconds, and incubate at 56°C for 10 minutes.

[0040] 3) Add 600 μL of lysis buffer, vortex mix for 30 seconds, and incubate at 70°C for 10 minutes.

[0041] 4) After centrifugation, add 400 μL of binding buffer and vortex for 30 seconds.

[0042] 5) Transfer 1440 μL of the mixture from the previous step to the adsorption column (in a 2 mL collection tube) in two portions without wetting the rim. Close the lid and centrifuge at 6000 x g for 1 min.

[0043] 6) Place the adsorption column in a clean 2 mL collection tube and discard the collection tube containing the filtrate. Close each adsorption column to avoid aerosol formation during centrifugation.

[0044] 7) Open the adsorption column and add 700 μL of Wash 1. Centrifuge at 6000 x g for 1 min. Place the adsorption column in a clean 2 mL collection tube and discard the collection tube containing the filtrate.

[0045] 8) Open the adsorption column, add 750 μL of Wash 2, and centrifuge at 6000×g for 1 min.

[0046] 9) Place the adsorption column in a new 2 mL collection tube and discard the old collection tube along with the filtrate. Centrifuge at full speed for 2 minutes.

[0047] 10) Place the adsorption column in a clean 1.5 mL elution tube and discard the collection tube. Carefully add 60 μL of elution buffer to the center of the column membrane. Cover the tube and incubate at room temperature for 1-5 minutes. Centrifuge at full speed for 1 minute. The liquid in the collection tube is the sample DNA to be tested. Use immediately or freeze at -20°C.

[0048] S2. Prepare PCR reaction system: The PCR reaction system includes a DNA template, a primer-probe combination targeting the target sequence, a reaction substrate, and a polymerase.

[0049] A digital PCR reaction premix (1 reaction) was prepared using the droplet digital PCR reaction premix according to the recipe in Table 2. The enzymes in Table 2 are DNA polymerases purchased from Beijing MicroRead Gene Technology Co., Ltd.

[0050] Table 2 Digital PCR reaction premix

[0051] Components volume 2× Digital PCR Master Mix 10 μL Enzyme (1500U / ml) 1.0μL Primer probe mixture (final concentration 10uM) 5μL Detection template 10ng Total volume Make up to 20 μL with nuclease-free water

[0052] The reaction solution was vortexed for 30 seconds, centrifuged briefly to collect the reaction solution at the bottom of the tube, and placed on ice for later use.

[0053] S4. Preparation of microdroplets

[0054] Samples were processed according to the operating instructions of the fully automatic sample processing system (Nanjing Puji Biomedical Co., Ltd., model: BMDG-50W-RV). The consumables involved included droplet detection oil (Nanjing Puji Biomedical Co., Ltd., catalog number: S02000101), punctureable heat-sealed film, PCR plates, pipettes (2 μl, 20 μL, 200 μL), DNase- and RNase-free centrifuge tubes, and DNase- and RNase-free pipette filter tips.

[0055] Place the droplet generation chip in the chip holder, add 50 μL of droplet generation oil to the oil phase well, add 20 μL of PCR reaction mix with template to the sample well, and then add 5 μL of sealant above the aqueous phase in each sample well. After the oil and aqueous phases are added, cover the droplet generation chip with the sealing gasket and place the droplet generation chip in the sample preparation instrument for droplet generation.

[0056] After the droplets are generated, carefully transfer the generated droplets (approximately 45-55 μL) to the PCR reaction plate in sequence;

[0057] After the droplets are transferred to the PCR reaction plate, cover it with punctureable heat-sealing film and place it on a preheated microplate sealer for sealing.

[0058] S6. Thermal Cycling Reaction

[0059] Place the 96-well plate in a nucleic acid amplification instrument and perform thermal cycling. The reaction program is shown in Table 3 below:

[0060] Table 3 Digital PCR reaction program

[0061]

[0062]

[0063] S6. Amplification was performed using a PCR amplifier (Nanjing Puji Biomedical Co., Ltd., model: BMCV96-RV). After amplification, the test results were analyzed using a biochip analyzer (Nanjing Puji Biomedical Co., Ltd., model: BMBC-610-RV).

[0064] S7. Place the PCR plate in a biochip analyzer for droplet detection, determine the copy number of the target gene amplified by the primer combination based on the detection results, and analyze the copy number information of the fragment to be tested in the sample based on the detection results.

[0065] Data analysis methods:

[0066] 1) This example establishes a method for detecting porcine, ovine, bovine, rabbit, and mouse-derived components in meat and meat products. Two genes from each species are selected for quantitative analysis. Primer probes are designed for the porcine ND5 / D-loop gene, the ovine ND5 / cytb gene, the bovine β-actin / Rd1 gene, the rabbit DQA / cytb gene, and the mouse 12S rDNA / ATP6 gene. Universal primers are designed for β-actin and 16S rDNA. Total DNA content is analyzed, and the differences between single-site and dual-site detection results are evaluated.

[0067] 2) The content of a certain animal-derived component is expressed by calculating the relative content of a certain animal-derived DNA in the sample. The quantitative analysis model is: p / % = a / t×100, where p is the content of pig-derived components in meat products (copy number) / %, a is the number of pig-derived DNA copies in the sample / ng, and t is the number of total DNA copies in the sample / ng.

[0068] Verify data:

[0069] The content analysis of different components in the known mixed meat products and the detection and analysis of the content of different animal-derived components in the muscle tissue of the meat products to be tested (Note: the total meat content in the meat products to be tested is about 70%), it is known that the test results of the mixed meat products are similar to the mixing ratio, and the meat components of the meat products to be tested not only contain beef tissue, but also other meat components such as pork and mutton.

[0070] Table 4 Test results

[0071]

[0072] This example establishes a complete method for analyzing animal-derived ingredients in food, which has the following advantages over existing detection methods:

[0073] 1) Timeliness and simplicity: The digital PCR method of the present invention requires only three steps: droplet generation, amplification, and detection, and can be completed in one day. The method is convenient to operate, and the analysis algorithm is simple and easy to use.

[0074] 2) Economical: This method allows for the simultaneous analysis of multiple target components, offering greater time and cost-effectiveness. Sensitivity and Accuracy: Digital PCR offers higher sensitivity than fluorescent PCR. For complex food components, digital PCR is highly tolerant to inhibitors, and test results are unaffected by PCR efficiency and varying food processing techniques. Analysis of test results does not require the creation of a standard curve, allowing for convenient absolute quantification.

[0075] 3) Stability: This paper establishes a method for detecting and analyzing the copy number of different animal-derived ingredients. Based on the experience of previous analyses, it provides judgment criteria for evaluating slight differences in gene copy number of different animal-derived ingredients, and the feasibility of this method has been verified.

[0076] Example 2 Foodborne Microbial Detection

[0077] This embodiment designs a method for simultaneous quantitative detection of multiple pathogenic bacteria DNA in food.

[0078] The primer and probe design scheme is as follows:

[0079] Table 5 Primer design

[0080]

[0081]

[0082] In Table 5, FAM, BHQ1, CY5, BHQ2, VIC, ROX, CY7, BHQ3, and CY5.5 are all conventional fluorescent groups.

[0083] The experimental steps are as follows:

[0084] S1. DNA extraction from samples: After homogenization, the samples were ground and then DNA was extracted. For specific methods, refer to the instructions of the Nucleic Acid Extraction Kit (Spindle Column Method) (Puji Biotechnology).

[0085] S2. Preparation of PCR reaction system: The PCR reaction system includes a DNA template, a primer-probe combination targeting the target sequence, a reaction substrate and a polymerase (Beijing MicroRead Gene Technology Co., Ltd.).

[0086] Table 6 Digital PCR reaction premix

[0087] Components volume 2× Digital PCR Master Mix 10 μL Enzyme (1500U / ml) 1.0μL Primer probe mixture (final concentration 10uM) 5μL Detection template 10ng Total volume Make up to 20 μL with nuclease-free water

[0088] S3. Add the PCR reaction system and droplet generation oil to the droplet generation chip, and place the droplet generation chip in a fully automatic sample processing system (Nanjing Puji Biomedical Co., Ltd., model: BMDG-50W-RV) for droplet generation. After droplet generation is completed, the droplets are transferred to a PCR plate and sealed.

[0089] S4. Use PCR amplification instrument (Nanjing Puji Biomedical Co., Ltd., model: BMCV96-RV) for amplification.

[0090] S5. After the PCR amplification reaction is completed, the test results are analyzed using a biochip analyzer (Nanjing Puji Biomedical Co., Ltd., model: BMBC-610-RV).

[0091] S6. Analyze the copy number information of the fragment to be tested in the sample according to the detection result.

[0092] Data analysis methods:

[0093] 1) This example establishes a detection method for foodborne pathogens, including Listeria monocytogenes (hly, prfA, mpl), Salmonella (invA, tcps, hilA), Vibrio parahaemolyticus (tdh, trh, irgB), Staphylococcus aureus (nuc, sec, coa), Shiga toxin-producing Escherichia coli (stx1, stx2, Z3276), and Bacillus cereus (nheA, bceT, hblA). Primers and probes are designed for each of the microorganisms to be tested.

[0094] 2) Analysis of target sequences was performed by comparing the results of single-gene detection with the average of the three-gene detection results of the same genus to analyze the reliability and quantitative differences of the detection results.

[0095] 3) Verify data:

[0096] Analysis of the detection sensitivity of different microbial reference products, comparison of the detection results of amplification by one pair of primers and simultaneous amplification by three pairs of primers:

[0097] Table 7

[0098]

[0099] a) The DNA content of the test bacteria was tested in fresh milk cake mixed with the test bacteria DNA, and the detection ability of this method for microorganisms in food was analyzed. After the microbial DNA was extracted, the test result showed that it was about 70% of the mixed DNA content.

[0100] Table 8

[0101]

[0102] *Total number of droplets is used as a quality control indicator to evaluate the quality of the test results

[0103] a) Fresh milk cakes that had been stored at room temperature (20°C) for one week were tested to analyze the ability to detect foodborne microorganisms. The results are as follows:

[0104] Table 9

[0105]

[0106]

[0107] This example establishes a foodborne microbial detection method, which has the following advantages over existing detection methods:

[0108] Compared with the prior art, the present invention has the following beneficial effects:

[0109] Timeliness and simplicity: Compared with traditional culture methods, the present invention does not require the cultivation of microorganisms, is easy to operate, and can complete detection and analysis within 1 day.

[0110] Economical: Compared with existing fluorescent PCR products, the present invention can detect all the required pathogens at one time, achieving the purpose of "one tube" detection and feedback of multiple pathogen content analysis, saving consumables and labor costs.

[0111] Reliable results: The digital PCR technology used in this invention has high detection sensitivity and can effectively detect pathogens as low as 10 copies / reaction, even when the microbial content is low.

[0112] Stability: This method establishes a method for the detection of pathogenic bacteria in food that can simultaneously detect multiple pathogens in one reaction, and evaluates the minimum detection limit for each pathogen, which can more reasonably evaluate the test results.

[0113] Example 3 Analysis of Abnormal Y Chromosome Copy Number in Males

[0114] The present application provides a method for analyzing Y chromosome copy number abnormalities. 30 detection sites are designed for the Y chromosome (including 15 STS sites in the AZF region) and chromosome 1, respectively, and chromosome 1 is used as an internal reference to evaluate Y chromosome copy number variation.

[0115] The primer and probe design scheme is as follows:

[0116] Table 10

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] In Table 10, FAM, BHQ1, and VIC are all conventional fluorescent groups.

[0123] The experimental steps are as follows:

[0124] S1. DNA extraction from peripheral blood samples: Blood samples can be directly used for DNA extraction without grinding. The extraction method refers to the instructions of the Nucleic Acid Extraction Kit (Centrifugal Column Method) (Puji Biotechnology).

[0125] S2. Prepare PCR reaction system: The PCR reaction system includes a DNA template, a primer-probe combination targeting the target sequence, a reaction substrate, and a polymerase. The reaction system is the same as Table 2 and Table 3 of Example 2.

[0126] S3. Add the PCR reaction system and droplet generation oil to the droplet generation chip, and place the droplet generation chip in a fully automatic sample processing system (Nanjing Puji Biomedical Co., Ltd., model: BMDG-50W-RV) for droplet generation. After droplet generation is completed, the droplets are transferred to a PCR plate and sealed.

[0127] S4. Amplification was performed using a PCR amplifier (Nanjing Puji Biomedical Co., Ltd., model: BMCV96-RV).

[0128] S5. After the PCR amplification reaction was completed, the test results were analyzed using a biochip analyzer (Nanjing Puji Biomedical Co., Ltd., model: BMBC-610-RV).

[0129] S6. Analyze the copy number information of the fragment to be tested in the sample according to the detection result.

[0130] Data Analysis:

[0131] (1) Using chromosome 1 as the internal reference, we analyzed the copy number variation of the Y chromosome. We set 30 detection sites on chromosome 1 and chromosome Y, respectively. Fifteen of the detection sites on chromosome Y were located in the AZF region, which is a high-frequency region for Y chromosome microdeletions. The CCN ratio of the detected Y chromosome to the reference chromosome 1 was calculated, i.e., RChrY / Chr1, using the following formula:

[0132]

[0133] In formula (1), R represents ratio, CCN represents chromosome copy number; the theoretical value of the ratio of chromosome Y with chromosome 1 as the reference (2), RChrY / Chr1, is approximately equal to 0.5. The confidence interval of this value in the normal population is determined through calibration with clinical samples. If it is less than or greater than this interval, the specific copy number variation type needs to be reviewed.

[0134] Verify data:

[0135] ① Correlation analysis of the effects of different primer pairs on Y chromosome detection results. Increasing the number of primer pairs can improve the stability of the detection results. The correlation between the number of primer pairs and the accuracy of Y chromosome ratio measurement is as follows: Figure 1 shown.

[0136] ②Comparison of the results of Y chromosome detection with 5ng and 10ng sample loading, samples as low as 5ng can be effectively detected, and the test results are as follows Figure 2 shown.

[0137] This example provides a method for analyzing abnormalities in the Y chromosome copy number in men, which has the following advantages over existing technologies:

[0138] (1) Timeliness and simplicity: The digital PCR of the present invention only requires three steps: droplet generation, amplification, and detection, and qualitative and quantitative detection can be completed in one day. The operation method of the present invention is convenient, the analysis algorithm is simple, and it is easy to use.

[0139] (2) Economical: This method is based on PCR technology and is relatively cost-effective compared to more complex detection technologies such as NGS. For samples with a risk of Y chromosome mutation, high-risk samples requiring review can be subdivided, reducing clinical testing costs and the risk of over-testing.

[0140] (3) Sensitivity and accuracy: Based on the high sensitivity of digital PCR, the present invention can detect small differences in gene copy number variations and can effectively analyze them. By increasing the number of detection sites, the accuracy of detection can be improved.

[0141] Stability: It has been verified that the detection range designed by the present invention can meet the analysis of Y staining copy number abnormalities. The quantitative analysis has high sensitivity and accuracy, the analysis method is simple, the operation method is concise and efficient, and the stability of laboratory operations is improved.

Claims

1. A method for quantitatively detecting slight differences in the copy number of a specific DNA sequence, characterized in that: The specific steps are as follows: Extract sample DNA as a detection template; 2) Design PCR primers and probes for each target locus and prepare a digital PCR reaction master mix: 10 μL of 2× digital PCR master mix, 1.0 μL of 1500 U / mL enzyme, 5 μL of a primer-probe mixture with a final concentration of 10 μM, 10 ng of detection template, and make up to 20 μL with nuclease-free water. 3) Preparation of droplets Using a fully automated sample processing system, add 20 μL of the digital PCR reaction mix prepared in step 2) to generate droplets. 4) PCR reaction Placing the droplets obtained in step 3) in a nucleic acid amplification instrument for PCR reaction to obtain amplified products; The PCR reaction program was as follows: step 1: 25°C, reaction for 10 min, 1 cycle; step 2: 95°C, reaction for 10 min, 1 cycle; step 3: 95°C for 10 s, 62°C for 50 s, 55 cycles; step 4: 98°C for 10 min, 1 cycle; step 5: 16°C for 1 cycle; 5) Detect the amplified product using a biochip analyzer to determine the copy number of the target gene amplified by the primer combination.

2. A PCR primer set for animal-derived component analysis, characterized in that: The primer set includes primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ IN NO.36; wherein, the 5' ends of primers SEQ IN NO.3 and SEQ IN NO.6 are both connected to a FAM fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.9 and SEQ IN NO.12 are both connected to a VIC fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.15 and SEQ IN NO.18 are both connected to a ROX fluorescent group, and the 3' ends are both connected to a BHQ2 fluorescent group; the 5' ends of primers SEQ IN NO.21 and SEQ IN NO.24 are both connected to a CY5 fluorescent group, and the 3' ends are both connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.27 and SEQ IN NO.30 are both connected to a CY5.5 fluorescent group, and the 3' ends are both connected to a BHQ3 fluorescent group; the 5' ends of primers SEQ IN NO.33 and SEQ IN The 5' end of NO.36 is connected to the CY7 fluorescent group, and the 3' end is connected to the BHQ3 fluorescent group.

3. A PCR primer set for detecting foodborne pathogens, characterized in that: The primer set includes primers with nucleotide sequences as shown in SEQ ID NO.37 to SEQ IN NO.90; wherein, the 5' ends of primers SEQ IN NO.39, SEQ IN NO.42, and SEQ IN NO.45 are all connected to a FAM fluorescent group, and the 3' ends are all connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.48, SEQ IN NO.51, and SEQ IN NO.54 are all connected to a CY5 fluorescent group, and the 3' ends are all connected to a BHQ2 fluorescent group; the 5' ends of primers SEQ IN NO.57, SEQ IN NO.60, and SEQ IN NO.63 are all connected to a VIC fluorescent group, and the 3' ends are all connected to a BHQ1 fluorescent group; the 5' ends of primers SEQ IN NO.66, SEQ IN NO.69, and SEQ IN NO.72 are all connected to a ROX fluorescent group, and the 3' ends are all connected to a BHQ2 fluorescent group; the 5' ends of primers SEQ IN NO.75, SEQ IN NO.78, and SEQ IN NO. The 5' end of primers NO.81 was connected to the CY7 fluorescent group, and the 3' end was connected to the BHQ3 fluorescent group; the 5' end of primers SEQ IN NO.84, SEQ IN NO.87, and SEQ IN NO.90 were connected to the CY5.5 fluorescent group, and the 3' end was connected to the BHQ3 fluorescent group.

4. A PCR primer set for detecting the copy number of the male Y chromosome, characterized in that the primer set comprises primers with nucleotide sequences as shown in SEQ ID NO.91 to SEQ ID NO.269; wherein, The 5'-ends of the primers SEQ IN NO.93, SEQ IN NO.96, SEQ IN NO.99, SEQ IN NO.102, SEQ IN NO.105, SEQ IN NO.108, SEQ IN NO.110, SEQ IN NO.113, SEQ IN NO.116, SEQ IN NO.119, SEQ IN NO.122, SEQ IN NO.125, SEQ IN NO.128, SEQ IN NO.131, SEQ IN NO.134, SEQ IN NO.137, SEQ IN NO.140, SEQ IN NO.143, SEQ IN NO.146, SEQ IN NO.149, SEQ IN NO.152, SEQ IN NO.155, SEQ IN NO.158, SEQ IN NO.161, SEQ IN NO.164, SEQ IN NO.167, SEQ IN NO.170, SEQ IN NO.173, SEQ IN NO.176, SEQ IN NO.179 are all linked with VIC fluorophores at the 5'-ends and BHQ1 fluorophores at the 3'-ends; the 5'-ends of the primers SEQ IN NO.182, SEQ IN NO.185, SEQ IN NO.188, SEQ IN NO. 191, SEQ IN NO.194, SEQ IN NO.197, SEQ IN NO.200, SEQ IN NO.203, SEQ IN NO.206, SEQ IN NO.209, SEQ IN NO.212, SEQ IN NO.215, SEQ IN NO.218, SEQ IN NO.221, SEQ IN NO.224, SEQ IN NO.227, SEQ IN NO.230, SEQ IN NO.233, SEQ IN NO.236, SEQ IN NO.239, SEQ IN NO.242, SEQ IN NO.245, SEQ IN NO.248, SEQ IN NO.251, SEQ IN NO.254, SEQ IN NO.257, SEQ IN NO.260, SEQ IN NO.263, SEQ IN NO.266, SEQ IN NO.269 are all linked with FAM fluorophores at the 5'-ends and BHQ1 fluorophores at the 3'-ends.

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