Poultry meat campylobacter quantitative detection method based on microdroplet type digital PCR and application of poultry meat campylobacter quantitative detection method
By using droplet digital PCR technology, specific primer-probe combinations were designed and reaction conditions were optimized to solve the problems of insufficient sensitivity and stability in Campylobacter detection in poultry meat, achieve highly sensitive, interference-resistant absolute quantitative detection, and improve detection accuracy and stability.
Patent Information
- Application Number
- CN202510964827.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-09-19
AI Technical Summary
Existing methods for detecting Campylobacter in poultry meat lack sensitivity and are difficult to effectively achieve absolute quantification. Traditional methods are time-consuming, and fluorescent quantitative detection is easily affected by amplification efficiency inhibition and standard curve deviation. There is a lack of specific primers and pretreatment solutions for Campylobacter in chicken, resulting in low detection limits and poor stability.
Using droplet digital PCR technology, specific primer-probe combinations (including forward primers, reverse primers, and probes) were designed and reaction conditions were optimized. Absolute counts of target nucleic acids were achieved using the Poisson distribution principle. Combined with 2×dPCR Probe Master Mix Plus and droplet-generating oil, a Campylobacter detection kit was developed for highly sensitive, interference-resistant quantitative detection.
It has achieved highly sensitive and interference-resistant quantitative detection of Campylobacter in chicken, with the detection limit exceeding 1000 CFU/g and the intra-batch/inter-batch coefficient of variation both less than 15%, which is significantly better than traditional methods. The positive detection rate of clinical samples has increased by 42.8%.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for quantitatively detecting Campylobacter in poultry meat based on droplet digital PCR and an application thereof. Background Art
[0002] Foodborne pathogens are a major threat to food safety and public health. Campylobacter is a major pathogen causing bacterial gastroenteritis. According to the World Health Organization, Campylobacter infection has become the leading cause of bacterial foodborne illness, with common sources of contamination including poultry, dairy products, and untreated drinking water. Chicken and its products are important vectors for Campylobacter transmission. Consuming contaminated food can lead to diarrhea, fever, and even serious complications such as Guillain-Barré syndrome. Therefore, establishing rapid and accurate methods for detecting Campylobacter is crucial for food safety monitoring and disease prevention and control.
[0003] Currently, traditional methods for detecting Campylobacter rely primarily on culture, enzyme-linked immunosorbent assay (ELISA), and real-time fluorescence quantitative PCR (qPCR). While culture is the gold standard, it is time-consuming and its sensitivity is limited by the viability of the bacteria. While fluorescence quantitative detection significantly improves detection efficiency, its quantitative results are susceptible to amplification efficiency inhibition and standard curve deviation, making absolute quantification difficult to achieve. In recent years, droplet digital PCR (ddPCR) has demonstrated significant potential in pathogen detection due to its high sensitivity, absolute quantification capability, and strong resistance to inhibitors. This technology, which partitions the reaction system into thousands to millions of droplets and utilizes the Poisson distribution principle to achieve absolute counts of target nucleic acids, eliminates the need for standard curves and is particularly suitable for the precise detection of low-abundance pathogens. Previous studies have demonstrated the superiority of ddPCR in detecting bacteria, viruses, and drug-resistance genes. However, the lack of specific primers and pretreatment protocols for Campylobacter in chicken results in low detection limits and poor stability. Furthermore, its application in the quantitative detection of Campylobacter in poultry meat has not been systematically established. Therefore, the development of a rapid ddPCR-based method for the quantitative detection of Campylobacter in chicken is of great significance for improving the accuracy and stability of detection. Summary of the Invention
[0004] The present invention aims to provide a method and application for the quantitative detection of Campylobacter in poultry meat based on droplet digital PCR to address the aforementioned issues with the prior art. The primer-probe combination and detection method provided by the present invention enable highly sensitive, interference-resistant quantitative detection of Campylobacter in chicken meat.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The present invention provides a primer-probe combination for detecting Campylobacter, comprising a forward primer as shown in SEQ ID NO.7, a reverse primer as shown in SEQ ID NO.8 and a probe as shown in SEQ ID NO.9.
[0007] Furthermore, the 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quenching group.
[0008] The present invention also provides the use of the primer-probe combination in preparing a product for detecting Campylobacter.
[0009] Furthermore, the product includes a detection reagent or a kit.
[0010] The present invention also provides a Campylobacter detection kit, comprising the primer-probe combination.
[0011] Furthermore, the Campylobacter detection kit also contains 2×dPCRProbe Master Mix Plus and droplet generation oil.
[0012] The present invention also provides the use of the Campylobacter detection kit in poultry food safety testing.
[0013] The present invention provides a method for detecting Campylobacter for non-diagnostic purposes, comprising the following steps: using the DNA of a sample to be tested as a template, performing a ddPCR reaction using the above-mentioned primer-probe combination, and calculating the Campylobacter copy number based on the droplet signal.
[0014] The droplet signal, the raw output of the ddPCR assay, is converted to the absolute copy number of the target DNA using a Poisson distribution model. This is then converted to the Campylobacter CFU / g value based on pre-processing parameters. Through quantitative analysis using this method, a target value ≥1000 CFU / g indicates a product is at risk of Campylobacter contamination, a value that is directly linked to food safety and can contribute to foodborne illness.
[0015] Furthermore, the reaction system of the ddPCR reaction includes: the forward primer, reverse primer and probe, 2×dPCR Probe Master Mix Plus, and a DNA template; the concentrations of the forward primer and reverse primer are both 500 nmol / L; and the concentration of the probe is 250 nmol / L.
[0016] Furthermore, the reaction procedure of the ddPCR reaction was as follows: droplet stabilization at 60°C for 5 minutes; enzyme activation at 95°C for 5 minutes; denaturation at 95°C for 10 seconds, and annealing / extension at 58°C for 30 seconds, for a total of 40 cycles.
[0017] The present invention discloses the following technical effects:
[0018] By optimizing primer probes (SEQ ID NO. 7-SEQ ID NO. 9) and an annealing temperature of 58°C, the present invention achieves highly sensitive, interference-resistant quantitative detection of Campylobacter in chicken. The actual sample detection limit exceeded 1000 CFU / g, with both intra- and inter-batch coefficients of variation less than 15%. The anti-interference capability was significantly superior to traditional methods, and the positive detection rate of clinical samples increased by 42.8% compared to the culture method. The droplet digital PCR-based quantitative detection method for Campylobacter in poultry meat provided by the present invention provides reliable technical support for early monitoring of poultry meat safety risks. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0020] Figure 1 For screening Campylobacter primers by common PCR method; where M: DL 1000Marker; 1: 16S rRNA-1 primer, 2: 16S rRNA-2 primer; 3: 16S rRNA-3 primer; N: negative template;
[0021] Figure 2 Screening of Campylobacter primers and probes for ddPCR; 1: 16S rRNA-1 primer + probe + template 1; 2: 16S rRNA-1 primer + probe + template 2; 3: 16S rRNA-1 primer + probe + negative template; 4: 16S rRNA-3 primer + probe + template 1; 5: 16S rRNA-3 primer + probe + template 2; 6: 16S rRNA-3 primer + probe + negative template;
[0022] Figure 3 ddPCR specificity test results; 1: negative template; 2: positive template; 3: Campylobacter jejuni clinical isolate; 4: Campylobacter coli clinical isolate; 5: Salmonella typhimurium; 6: Escherichia coli; 7: Staphylococcus aureus; 8: Listeria monocytogenes; 9: Escherichia coli clinical isolate; 10: Enterococcus faecalis clinical isolate;
[0023] Figure 4 Amplification graphs for each serial dilution. DETAILED DESCRIPTION
[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.
[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0029] Example 1
[0030] 1 Materials and Methods
[0031] 1.1 Strains and DNA extraction
[0032] The test strains are listed in Table 1. Genomic DNA from standard strains 1-5 listed in Table 1 was extracted according to the instructions of the Bacterial Genome Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) and eluted with 50 μL of TE buffer. DNA from clinical isolates 6-9 was previously frozen in the Quality, Safety, and Processing Laboratory of the Jiangsu Poultry Science Research Institute.
[0033] Table 1 Test strains
[0034]
[0035]
[0036] 1.2 Materials and Reagents
[0037] 2×dPCR Probe Master Mix Plus (Cy5.5), Sniper digital PCR consumables, and droplet generation oil were purchased from Suzhou Sinafu Medical Technology Co., Ltd.; 2×Taq Master Mix (Dye Plus) was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; microbial culture media were purchased from Qingdao Haibo Biotechnology Co., Ltd.; and chicken samples were purchased from a local supermarket.
[0038] 1.3 Main instruments and equipment
[0039] Droplet digital PCR (DQ24, Suzhou Sinafu Medical Technology Co., Ltd.); ultramicro-volume UV spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, USA); biochemical incubator (HPS, Taicang Huamei Biochemical Instrument Factory); shaking incubator (ZQTY-70, Shanghai Zhichu Instrument Co., Ltd.); gel imaging system (JY04S-3E, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.); electrophoresis instrument (DYY-12, Beijing Liuyi Biological Co., Ltd.).
[0040] 1.4 Design and screening of primers and probes
[0041] The Campylobacter 16S rRNA gene (GenBank no. AH000014.2) was selected as the target sequence, and three pairs of primers and probes were designed for screening. The fluorescent group FAM was added to the 5' end of the probe, and the corresponding quencher group BHQ1 was added to the 3' end, as shown in Table 2. The primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The designed and synthesized primers were initially screened using conventional PCR. The effectiveness of these primers and probes was then verified using ddPCR, and the reaction conditions were optimized.
[0042] Table 2 PCR reaction primer and probe sequences
[0043]
[0044] 1.5 Establishment of ddPCR detection system and optimization of conditions
[0045] The standard PCR preliminary screening reaction system consisted of 12.5 μL of 2× Extaq Mix, 1 μL of each upstream and downstream primer (10 μmol / L), 1 μL of template DNA, and double-distilled water to a total of 25 μL. The reaction conditions were: 95°C denaturation for 1 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; and 10 min of extension at 72°C. PCR products were visualized by electrophoresis on a 1.5% agarose gel.
[0046] The ddPCR system was set up with 11 μL of 2× dPCR Probe Master Mix Plus, 1 μL of each upstream and downstream primer (10 μmol / L) (final concentration 500 nmol / L), 0.5 μL of probe (final concentration 250 nmol / L), 1 μL of DNA template, and double-distilled water to a total of 22 μL. The temperature-controlled reaction program was as follows: 60°C droplet stabilization for 5 min; 95°C enzyme activation for 5 min; 95°C denaturation for 10 s, 60°C annealing / extension for 30 s, for a total of 40 cycles. After the amplification reaction, the results were analyzed using the number of control droplets, uncertainty (95% CI), and 1D scatter plots in the run program analysis module.
[0047] Temperature optimization: Using Campylobacter ATCC 33560 genomic DNA as a template, ddPCR amplification reactions were performed at annealing temperatures of 52°C, 54°C, 56°C, 58°C, and 60°C, respectively. The optimal annealing temperature was determined based on the amplification results.
[0048] 1.6 Specificity and sensitivity of detection
[0049] The concentration of Campylobacter jejuni ATCC 33560 genomic DNA was determined by Nanodrop 2000 and was 100 ng / mL (5.7×10 7 Copies / μL), and diluted it to 1.0×10 7 Copies / μL and then dilute 10-fold to 1.0×10 0 Make copies / μL for later use.
[0050] Specificity test: The bacterial nucleic acids listed in Table 1 were detected using the optimized Campylobacter ddPCR method to verify the specificity of the method.
[0051] Sensitivity test: Diluted Campylobacter ATCC 33560 genomic DNA was subjected to ddPCR detection. Three parallel tests were performed at each concentration. A standard curve was established and the correlation coefficient was calculated.
[0052] 1.7 Artificial pollution test
[0053] Sterile sample preparation: Pseudo-sterile samples were prepared and validated according to the method of Chen Lan (Screening, identification, and rapid detection of metabolic markers for three foodborne pathogens in chicken [D]. Yangzhou University, 2024. DOI: 10.27441 / d.cnki.gyzdu.2024.000076). Total colony counts were determined according to the National Food Safety Standard for Microbiological Examination of Foods—Determination of Total Colony Counts (GB 4789.2-2022).
[0054] Campylobacter was cultured in MH Broth and cultured at 42°C in a microaerophilic (5% O2, 10% CO2 and 85% N2) atmosphere for 36 h before measuring the OD value of the bacterial suspension. 600 , adjust the concentration of bacterial suspension to an absorbance of 0.7, at which point the bacterial suspension concentration is 1×10 7 CFU / mL, the bacterial suspension was diluted 10-fold to obtain 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 2 CFU / mL, 10 0 CFU / mL bacterial solution. A pseudo-sterile sample was selected as the added matrix. 25g of pseudo-sterile chicken sample was added with 2mL of sterile distilled water as a negative control. 2mL of different gradient bacterial solutions were drawn and added to 25g of pseudo-sterile chicken matrix, with each addition amount added in triplicate.
[0055] 1.8 Sample pretreatment
[0056] The sample was added to 100 mL of PBS solution and beat with a homogenizer for 2 minutes. 1.5 mL of the sample solution of each dilution was collected in a 2 mL centrifuge tube and centrifuged at 12000 g for 2 minutes. The precipitate was collected and the bacterial genomic DNA was extracted using a kit method. The precipitate was finally dissolved in 50 μL of LTE buffer and tested by ddPCR. The content of target bacteria in the sample (CFU / g) was calculated according to the following formula.
[0057]
[0058] Note: C is the target bacterial content in the test sample (CFU / g); N is the nucleic acid copy number measured in 22 μL ddPCR reaction system (Copies / 22 μL); V1 is the volume of the eluent during nucleic acid extraction (μL); V2 is the volume of the template DNA in the ddPCR system (μL); V3 is the volume of the sample solution during nucleic acid extraction (μL); f is the dilution factor of the sample when added to the PBS solution.
[0059] Stability test: Contaminated bacterial solutions of different concentrations were selected for testing. Three replicate wells were set for each concentration, and the standard deviation and coefficient of variation were calculated to evaluate the intra-batch stability of the method. Three independent tests were performed under the same reaction conditions, and the standard deviation and coefficient of variation were calculated to evaluate the inter-batch stability of the method.
[0060] 1.9 Clinical Sample Testing
[0061] Fifty samples of commercially available chicken products (including chilled chicken, chicken breast, chicken legs, diced chicken, etc.) were collected. The 50 samples were identified and detected using traditional bacterial isolation and culture methods and the Campylobacter ddPCR method established in the present invention, and the compliance rate of the two methods was evaluated.
[0062] 2 Results and Analysis
[0063] 2.1 Establishment and optimization of Campylobacter ddPCR method
[0064] The three primer pairs were tested by conventional PCR and found that 16s rRNA-1 and 16s rRNA-3 could amplify products, but primer 16s rRNA-2 was not effectively amplified ( Figure 1 ). 16s rRNA-1 and 16s rRNA-3 were amplified by ddPCR. The results showed that under the same template, the fluorescence signal of the 16s rRNA-3 probe was stronger and the negative discrimination was better ( Figure 2 ), droplet efficiency is higher. Among the six annealing temperatures, 58°C annealing performed better than the others. Therefore, we subsequently selected a 16srRNA-3 primer probe and a 58°C annealing temperature to establish a ddPCR reaction system.
[0065] 2.2 Specificity and sensitivity
[0066] The 16s rRNA-3 primer probe was used to perform ddPCR specificity verification on Salmonella typhimurium, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes from ATCC, 20 Campylobacter jejuni, 20 Campylobacter coli, 20 Escherichia coli and 20 Enterococcus faecalis from clinical isolates. The results showed that 20 Campylobacter jejuni and 20 Campylobacter coli showed positive amplification signals, while the test results of other Enterobacteriaceae strains were negative ( Figure 3 ), demonstrating that the 16s rRNA-3 primer probe has good specificity and accuracy.
[0067] 7 gradient nucleic acid concentrations from 1×10 6 Copies / μL decreased to 1×10 0 Copies / μL, gradient dilution amplification diagram as shown Figure 4 As shown in the figure, the method can also detect when the detection concentration is lower than 10 copies. With the logarithm of the dilution gradient (Log) as the horizontal axis and the logarithm of the copy number (Log) as the vertical axis, the standard curve of ddPCR detection of Campylobacter is y=0.437x+0.7608, R 2 =0.9927, showing a good linear relationship.
[0068] 2.3 Detection of artificially contaminated samples
[0069] As shown in Table 3, artificially contaminated positive samples were generated by spiking pseudosterile chicken matrix with a gradient dilution of Campylobacter (ATCC 33560). The results demonstrate excellent quantitative performance using ddPCR, with a limit of detection of 1000 CFU / g for actual contaminated samples. The maximum deviation across the eight spiking levels was 14.9%, and the coefficient of variation for triplicate assays within each concentration batch was less than 15%. Three independent tests conducted under the same conditions revealed inter-assay coefficients of variation of no more than 15%, demonstrating the good inter-assay reproducibility and robust test results of the ddPCR method.
[0070] Table 3 Quantitative results of ddPCR detection of Campylobacter in artificially contaminated chicken samples
[0071]
[0072] 2.4 Clinical sample testing
[0073] The bacterial culture method (National Food Safety Standard Food Microbiology Test Total Colony Count Determination (GB4789.2-2022)) and the Campylobacter ddPCR method established by the present invention were used to identify and detect 50 chicken products. The results showed that the number of positive samples detected by the bacterial culture method was 7 strains, with a positive rate of 14%, and the number of positive samples detected by ddPCR was 10 strains, with a positive rate of 20%. Among them, the 7 positive samples detected by ddPCR and the bacterial culture method were completely consistent. The target bacteria content in the other three positive samples was about 1200 CFU / g. The bacterial culture method may have inhibited the growth of target bacteria during the culture process. The above shows that the detection method established by the present invention can effectively detect clinical samples and can perform quantitative analysis.
[0074] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A primer-probe combination for detecting Campylobacter, characterized in that: It includes a forward primer as shown in SEQ ID NO.7, a reverse primer as shown in SEQ ID NO.8 and a probe as shown in SEQ ID NO.
9.
2. The primer-probe combination according to claim 1, characterized in that The 5' end of the probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quenching group.
3. Use of the primer-probe combination according to claim 1 or 2 in the preparation of a product for detecting Campylobacter.
4. The use according to claim 3, characterized in that The products include detection reagents or kits.
5. A Campylobacter detection kit, characterized in that: Comprising the primer-probe combination according to claim 1 or 2.
6. The Campylobacter detection kit according to claim 5, characterized in that The Campylobacter detection kit also contains 2×dPCR Probe Master Mix Plus and droplet generation oil.
7. Use of the Campylobacter detection kit according to claim 5 or 6 in poultry food safety testing.
8. A method for detecting Campylobacter for non-diagnostic purposes, characterized in that: The method comprises the following steps: using the DNA of a sample to be tested as a template, performing a ddPCR reaction using the primer-probe combination according to claim 1 or 2, and calculating the Campylobacter copy number based on the droplet signal.
9. The Campylobacter detection method according to claim 8, characterized in that: The reaction system of the ddPCR reaction comprises: the forward primer, reverse primer and probe described in claim 1, 2×dPCRProbe Master Mix Plus, and a DNA template; the concentrations of the forward primer and reverse primer are both 500 nmol / L; and the concentration of the probe is 250 nmol / L.
10. The Campylobacter detection method according to claim 8, characterized in that: The reaction procedure of the ddPCR reaction was as follows: droplet stabilization at 60°C for 5 min; enzyme activation at 95°C for 5 min; denaturation at 95°C for 10 s, and annealing / extension at 58°C for 30 s, for a total of 40 cycles.