A multiplex digital PCR detection method for Campylobacter, Salmonella and Staphylococcus aureus

Abstract

The purpose of the present invention is to provide a multiplex digital PCR detection method for Campylobacter, Salmonella and Staphylococcus aureus, so as to detect three pathogenic bacteria more conveniently, quickly and accurately through one detection reaction. The primer and probe set, wherein the primer pair used to detect Campylobacter, the sequences of its upstream and downstream primers are SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the probe is SEQ ID NO:3; the primer pair used to detect Salmonella, the sequences of its upstream and downstream primers are SEQ ID NO:4 and SEQ ID NO:5, and the sequence of the probe is SEQ ID NO:6; the primer pair used to detect Staphylococcus aureus, the sequences of its upstream and downstream primers are SEQ ID NO:7 and SEQ ID NO:8, and the sequence of the probe is SEQ ID NO:9.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection services, and in particular relates to a multiplex digital PCR detection method for Campylobacter, Salmonella and Staphylococcus aureus. Background Art

[0002] In recent years, safety incidents caused by various pathogenic microorganisms and parasites infecting humans during the slaughtering, processing and consumption of livestock and poultry have become an important factor affecting public health.

[0003] Foodborne gastrointestinal diseases caused by Campylobacter are a serious economic and public health burden facing humanity in the 21st century. Due to the lack of reported data, it is difficult to estimate the overall scale of the disease worldwide. However, it is estimated that campylobacteriosis accounts for 8.4% of all diarrheal diseases in the world. The number of cases reported in the European Union each year exceeds 200,000, but the actual number of cases may be as high as 9 million per year because many people do not go to the hospital for treatment. The economic losses caused by campylobacteriosis, including medical expenses and productivity, are estimated to exceed 2 billion euros each year. Molecular biological detection methods for Campylobacter include conventional PCR, real-time fluorescence quantitative PCR and other methods.

[0004] Salmonella is a common and important zoonotic pathogen. It can not only cause animal diseases such as pullorum, paratyphoid fever, and abortion, but also cause typhoid fever, sepsis, food poisoning, and gastroenteritis in humans. Among food poisoning cases around the world, many cases of poisoning caused by Salmonella account for the first place. As an important indicator for pathogenic bacteria detection, Salmonella is of great significance in public health, food hygiene, animal husbandry and veterinary medicine, and entry-exit inspection and quarantine. Therefore, it is particularly important to establish a rapid Salmonella detection method. At present, the detection methods of Salmonella include culture method, immunological method, and PCR method. The detection method of Salmonella specified in the national standard is microbial culture method, which has the disadvantages of long operation time, low specificity and sensitivity, and false positives are prone to occur in immunological methods due to nonspecific adsorption. The ordinary PCR method cannot perform quantitative analysis on samples. Therefore, it is necessary to establish a quantitative PCR method for Salmonella.

[0005] Staphylococcus aureus (hereinafter referred to as S. aureus) is a common foodborne pathogen. Eating food contaminated with S. aureus enterotoxin can cause food poisoning. As one of the important indicators for judging food microbial contamination, S. aureus is also the focus of food supervision and random inspections. National food standards have set limits on S. aureus in a variety of foods. It is of great significance to explore accurate and rapid quantitative detection methods for S. aureus. The national standard method for S. aureus testing uses the traditional culture method, which has many steps and cumbersome operations. It takes 48 hours from sample preparation to initial screening results, and 3 to 4 days to final confirmation and identification. Summary of the invention

[0006] The purpose of the present invention is to provide a multiplex digital PCR detection method for Campylobacter, Salmonella and Staphylococcus aureus, so as to more conveniently, quickly and accurately detect the three pathogens through one detection reaction.

[0007] The present invention first provides a multiplex digital PCR detection primer and probe set for detecting Campylobacter, Salmonella and Staphylococcus aureus, wherein the sequence information of the primer pair and probe for detecting Campylobacter is as follows:

[0008] Upstream primer: 5′-ACACACGTGCTACAATGGCATA-3′ (SEQ ID NO: 1), downstream primer: 5′-TGGGACATATTTTATAGATTTGCT-3′ (SEQ ID NO: 2),

[0009] Probe: 5′-TACAATGAGACGCAATACCGCGAGGT-3′ (SEQ ID NO: 3);

[0010] The sequence information of the primer pairs and probes used to detect Salmonella are as follows:

[0011] Upstream primer: 5′-CGAACGGCGAAGCGTACTGGAA-3′ (SEQ ID NO: 4),

[0012] Downstream primer: 5′-CGGCAATGGCGTCACCTTTGAT-3′ (SEQ ID NO: 5),

[0013] Probe: 5′-CTTTACGGYTCCTTTGACGGTGCGATGAAG-3′ (SEQ ID NO: 6);

[0014] The sequence information of the primer pairs and probes used to detect Staphylococcus aureus is as follows:

[0015] Upstream primer: 5′-AGTTCGTCAAGGCTGGGCTAAAGT-3′ (SEQ ID NO: 7),

[0016] Downstream primer: 5′-GAATCAGCGTTGTCTTCGCTCCAA-3′ (SEQ ID NO: 8),

[0017] Probe: 5′-TATAAACCTAACAATACACATGAACAACTT-3′ (SEQ ID NO: 9);

[0018] Furthermore, the 5′ end or 3′ end of the probe is coupled with fluorescein;

[0019] As a specific description of the embodiment, the probe for detecting Campylobacter has fluorescein VIC connected to the 5′ end and fluorescein MGB connected to the 3′ end;

[0020] The probe for detecting Salmonella has fluorescein FAM linked to its 5′ end and fluorescein BHQ1 linked to its 3′ end;

[0021] The probe for detecting Staphylococcus aureus has fluorescein CY5 linked to its 5′ end and fluorescein MGB linked to its 3′ end;

[0022] The primer and probe set provided by the present invention can be used to prepare products for detecting Campylobacter, Salmonella and Staphylococcus aureus;

[0023] The product described, as a specific description of the embodiment, is a multiplex digital PCR detection product.

[0024] In another aspect, the present invention provides a multiplex digital PCR detection method for detecting Campylobacter, Salmonella and Staphylococcus aureus, which uses the above-mentioned primer and probe set for detection.

[0025] In the method, the amount of primer used is 17 μmol / 15 μL, and the amount of probe used is 6 μmol / 15 μL.

[0026] Furthermore, the amplification program conditions of the PCR in the method described are 95°C for 10 min; 95°C for 30 s, 60°C for 1 min, 40 cycles; 25°C for 10 min.

[0027] The multiplex digital PCR detection method established by the present invention can directly quantify Campylobacter, Salmonella and Staphylococcus aureus in one reaction; compared with the general digital PCR method, it saves reagent and labor costs; and compared with the indirect quantification of the fluorescent quantitative PCR method, the measurement is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Specificity test results of multiplex ddPCR method Figure Ⅰ , among which A1 is Salmonella, A3 is Enterococcus, B3 is Clostridium perfringens, C3 is Streptococcus, D3 is Escherichia coli, A4 is Listeria monocytogenes, and B4 is Mycobacterium.

[0029] Figure 2 : Specificity test results of multiplex ddPCR method Figure II , among which E4 is Campylobacter, A1 is Enterococcus, B1 is Clostridium perfringens, C1 is Streptococcus, D1 is Escherichia coli, A2 is Listeria monocytogenes, and B2 is Mycobacterium.

[0030] Figure 3 : Specificity test results of multiplex ddPCR method Figure III , among which H4 is Staphylococcus aureus, C1 is Enterococcus, D1 is Clostridium perfringens, A2 is Streptococcus, B2 is Escherichia coli, C2 is Listeria monocytogenes, and D2 is Mycobacterium.

[0031] Figure 4 : Detection sensitivity diagram of Campylobacter, where G2, G1, C4, C3, and C2 are 10 3 , 10 2 , 10 1 , 10 0 , 10 - 1 copies / μL concentration gradient;

[0032] Figure 5 : Detection sensitivity diagram of Salmonella, where E4, F4, E3, F3, and E2 are 10 3 , 10 2 , 10 1 , 10 0 , 10 - 1 copies / μL concentration gradient.

[0033] Figure 6 : Detection sensitivity diagram of golden glucose bacteria, where H3, H2, H1, D4, and D3 are 10 3 , 10 2 , 10 1 , 10 0 , 10 -1 copies / μL concentration gradient. DETAILED DESCRIPTION

[0034] Droplet digital PCR (ddPCR) is a method that processes samples into droplets, detects each droplet one by one after PCR amplification, and interprets the droplets with fluorescent signals as 1 and those without fluorescent signals as 0. The starting copy number of the target molecule can be obtained based on the Poisson distribution principle and the number and ratio of positive droplets. This method is a new nucleic acid detection and quantification method developed on the basis of real-time fluorescence PCR. It does not rely on standard curves and reference samples, and can directly measure the absolute copy number of the target gene in the sample. It has been widely used in pathogenic microorganism detection, rare gene mutation detection, and identification of source ingredients in food. Many studies have shown that droplet digital PCR is more sensitive than real-time fluorescence PCR quantitative methods. The multiplex digital PCR detection method can detect multiple target fragments simultaneously in one reaction system. It has the specificity and sensitivity of single digital PCR, and is faster and more economical. The design requirements of multiplex digital PCR primer probes are similar to those of multiplex fluorescent quantitative PCR primer probes, that is, primers need to be designed for highly specific target fragments to avoid nonspecific amplification; appropriate fluorescent group combinations need to be selected to avoid wavelength overlap; attention should be paid to the complementarity between different primers to avoid dimer formation; and the Tm values ​​of different primers should be coordinated to avoid mutual influence on amplification.

[0035] The present invention designs and screens suitable primer probes and establishes a multiplex digital PCR detection method, which provides an effective technical means for the clinical rapid detection of three animal-derived pathogens, namely Campylobacter, Salmonella and Staphylococcus aureus.

[0036] The present invention is described in detail below in conjunction with embodiments and drawings.

[0037] Example 1: Establishment of a multiplex ddPCR diagnostic method

[0038] 1.1 Preparation of positive control

[0039] Cultivate Campylobacter, Salmonella and Staphylococcus aureus respectively, pick the corresponding colonies with an inoculation loop, place in a centrifuge tube containing 0.5 mL of physiological saline, vortex evenly, centrifuge at 12000 r / min for 5 min, and discard the supernatant. Add 0.5 mL of sterile water and vortex to mix, boil in 100°C boiling water for 10 min, take out and cool in an ice bath for 10 min, centrifuge at 12000 r / min for 5 min, take the supernatant, dilute it 1:1000, and use it as positive control for Campylobacter, Salmonella and Staphylococcus aureus, respectively, for the screening of primer pairs and probes and the optimization of digital PCR conditions.

[0040] 1.2 Design of primers and probes

[0041] The sequences of Campylobacter 16S RNA gene (Genebank: Y11763.1), Salmonella invA gene (GenBank: CP053579.1) and Staphylococcus aureus nuc gene (Genebank: EF529607.1) were downloaded from GenBank, and two sets of primers and probes were designed for each pathogen using PrimerPlex software.

[0042] The applicability of target genes and primer probes was analyzed. Taking the 16S RNA gene of Campylobacter as an example, the 16S rRNA gene sequences of 129 strains of Campylobacter, including Campylobacter coli, Campylobacter jejuni, and Campylobacter rubrum, were queried and downloaded, and the DNA star software was used to compare them to determine the conserved regions of the 16S rRNA gene sequences of 130 strains; the 16S rRNA genes of Arcobacter and Helicobacter, which are closely related to Campylobacter, were downloaded, and it was found that the homology between them was low (the homology with Arcobacter brucei (Genebank: NR_043035.1) was 86.01%, and the homology with Helicobacter pylori (Genebank: NR_044761.1) was 86.01%), and the sequences at the primer probe positions were different; the 16S rRNA genes of diarrheagenic Escherichia coli and Streptococcus were downloaded for comparison, and their homology was less than 80%, and the sequences at the primer probe positions were different.

[0043] The Salmonella invA gene, Staphylococcus aureus nuc gene and their primer probes were analyzed in a similar manner to ensure the specificity and applicability of the designed primer probes.

[0044] The designed primer and probe sequences are shown in Table 1.

[0045] Table 1: Designed primers and probes

[0046]

[0047] 1.3 Screening of primers and probes

[0048] After designing the detection primers and probes for Campylobacter, Salmonella and Staphylococcus aureus, it is necessary to screen the primer probe combination and screen the primer groups with similar amplification efficiency to avoid the mutual influence between primers. The primer probe sequences designed in Table 1 were combined to obtain 8 groups in total. ①: αⅠA; ②: αⅠB; ③: αⅡA; ④: αⅡB; ⑤: βⅠA; ⑥: βⅠB; ⑦: βⅡA; ⑧: βⅡB. Results After the β primer probe was combined with other primer probes, its amplification efficiency decreased significantly; the B primer probe would produce certain nonspecific amplification in the combination; both Ⅰ and Ⅱ primer probes could be amplified well in the combination, but the copy number of Ⅱ was slightly higher. The final combination was ③: αⅡA. After the final primer probe (see Table 2) sequence was determined, it was handed over to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

[0049] Table 2: Primers and probes used in multiplex digital PCR detection method

[0050]

[0051]

[0052] 1.4 Optimization of digital PCR reaction conditions

[0053] Optimize the ddPCR reaction, including the optimization of PCR reaction conditions (primer, probe concentration, annealing temperature), the linearity of the method and the determination of the minimum detection limit.

[0054] The 100 μmol / L primer concentration was set to 0.13 μL, 0.15 μL, 0.17 μL, and 0.19 μL, the 100 μmol / L probe concentration was set to 0.03 μL, 0.045 μL, 0.06 μL, and 0.075 μL, and the annealing temperature was set to 56°C, 58°C, 60°C, and 62°C. The optimization results were determined by comparing the detection copy number, droplet generation number, droplet distribution state, and droplet fluorescence signal intensity of ddPCR.

[0055] Results When the amount of 100 μmol / L primer was 0.17 μL and the amount of 100 μmol / L probe was 0.06 μL, ddPCR had the highest detection copy number, and the droplet distribution state and the intensity of the droplet fluorescence signal were also good. Therefore, the amount of primer was determined to be 0.17 μL and the amount of probe was 0.06 μL.

[0056] Four annealing temperatures were set at 56, 58, 60, and 62°C. As a result, all four temperature gradients had good droplet distribution, and when the annealing temperature was 60°C, it had lower background fluorescence and the highest measurement value. Therefore, the annealing temperature was set to 60°C.

[0057] 1.5 Establishment of droplet digital PCR method

[0058] Finally, a digital PCR method was established. Rox was diluted tenfold for reaction. The reaction system (15 μL) was: 5×Mix 3 μL, 1 / 10 Rox 1 μL, 3 upstream primers at 100 μmol / L, 0.17 μL of downstream primers, 0.06 μL of 3 probes at 100 μmol / L, 7.8 μL of water, and 2 μL of template. Mix well, take 14 μL and add it to the sample well of the chip, add 16 μL of blocking oil, cover the lid, and put the chip into the digital PCR instrument for amplification. The PCR amplification program conditions were 95℃10min; 95℃30s, 60℃1min, 40 cycles; 25℃10min, and read the value after the reaction. The original reading of the instrument multiplied by 7.5 is the number of copies (copies / μL) in the sample.

[0059] Example 2: Specificity test of multiplex digital PCR method

[0060] The DNA of Campylobacter, Staphylococcus aureus, Salmonella, Enterococcus, Clostridium perfringens, Escherichia coli, Listeria monocytogenes, and Mycobacterium were used as templates, and the method established in Example 1 was used to detect them to evaluate the specificity of the method. Results The DNA of Enterococcus, Clostridium perfringens, Escherichia coli, Listeria monocytogenes, Listeria monocytogenes, and Mycobacterium was detected by the multiplex digital PCR method. When the positive control was established, the detection results of the above bacteria were all negative. It was confirmed that the multiplex digital PCR method has good specificity when detecting these non-specific bacteria (see Figure 1 , Figure 2 and Figure 3 ).

[0061] Example 3: Determination of detection limit and quantification limit of digital PCR detection method

[0062] Five concentration gradients (10 -1 , 10 0 , 10 1 , 10 2 , 10 3 The positive controls of Campylobacter, Salmonella and Staphylococcus aureus (1000 copies / μL) were used as templates, and each gradient was tested three times. The multiplex ddPCR method was used to amplify the samples, and the detection limit and quantification limit of the method were evaluated.

[0063] Results The number of droplets generated by all ddPCR reactions was greater than 10,000 droplets, indicating that all reaction droplets were generated normally, ensuring the accuracy of subsequent quantitative analysis. The test results of Campylobacter, Salmonella and Staphylococcus aureus nucleic acid after gradient dilution are shown in Table 3 and Figure 4-Figure 6 According to the concentration Tk and the test results, calculate the S of each concentration. k r k 、w k ; Corresponding to Sk and T k Perform linear regression and calculate the intercept g and slope h; reject the choice of constant model and adopt the straight line model; fit the model R and calculate the values ​​of a and b; calculate YC according to the values ​​of g, a, n and k1 (obtained from the table); calculate LC according to the b value, and calculate the values ​​of IDE0, IDE1, IDE2, IDE3, etc. After calculation, Campylobacter nucleic acid IDE2 is approximately equal to IDE3, and its IDE is 2.78 copies / μL; Salmonella nucleic acid IDE2 is approximately equal to IDE3, and its IDE is 3.03 copies / μL; Staphylococcus aureus nucleic acid IDE2 is approximately equal to IDE3, and its IDE is 7.71 copies / μL;

[0064] Based on the g, h, and b calculated above, the Z value is approximately 10, and the IQE is calculated accordingly. 10％ The corrected IQEs for Campylobacter nucleic acid were 11.57 copies / μL, for Salmonella nucleic acid was 11.42 copies / μL, and for Staphylococcus aureus nucleic acid was 18.01 copies / μL.

[0065] Table 3: Results of multiplex digital PCR detection method for positive controls of different concentrations (unit: copies / μL)

[0066]

[0067] Note: is the mean value and S is the standard deviation.

[0068] Example 4: Repeatability test of multiplex digital PCR method

[0069] 10 4 The positive controls of Campylobacter, Salmonella and Staphylococcus aureus with different concentration gradients were mixed in equal proportions and then diluted to a concentration gradient of 10 for each DNA. 3 , 10 2 , 10 1 , using these three concentration gradients as templates, repeated multiplex PCR detection three times; repeatability test was performed once every 5 days, for a total of 3 times. The repeatability test results are shown in Tables 4 and 5. The intra-group coefficient of variation was 1.27% to 4.12%, and the inter-group coefficient of variation was 2.97% to 6.21%. This shows that the established multiplex digital PCR method has good repeatability and reproducibility, and the test results are stable and reliable.

[0070] Table 4: Results of intra-group repeatability test of multiplex digital PCR method (unit: copies / μL)

[0071]

[0072] Table 5: Results of the inter-group repeatability test of the multiplex digital PCR method (unit: copies / μL)

[0073]

[0074]

[0075] Example 5: Clinical sample validation

[0076] 15 samples of Campylobacter, Salmonella and Staphylococcus aureus positive samples and 20 samples of negative samples were taken. The nucleic acid in the positive samples was extracted using a DNA extraction kit and used as clinical sample nucleic acid for multiple ddPCR identification. Results The results of 15 samples of Campylobacter, Salmonella and Staphylococcus aureus positive samples were all positive, and the results of 20 samples of Campylobacter, Salmonella and Staphylococcus aureus negative samples were all negative. It was confirmed that the established multiple ddPCR method can be used for the detection of clinical samples.

Claims

1. A multiplex digital PCR detection product, characterized in that: The multiplex digital PCR detection product comprises primer pairs and probe sets for detecting Campylobacter, Salmonella and Staphylococcus aureus, and the compositions of the primer pairs and probe sets are as follows: the sequences of the upstream and downstream primers of the primer pair for detecting Campylobacter are shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the probe for detecting Campylobacter is shown in SEQ ID NO:3; A primer pair for detecting Salmonella, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO:4 and SEQ ID NO:5, and the sequence of the probe for detecting Salmonella is shown in SEQ ID NO:6; A primer pair for detecting Staphylococcus aureus, wherein the sequences of the upstream and downstream primers are shown in SEQ ID NO:7 and SEQ ID NO:8, and the sequence of the probe for detecting Staphylococcus aureus is shown in SEQ ID NO:9; The 5′ end of the probe for detecting Campylobacter is connected to fluorescein VIC, and the 3′ end is connected to fluorescein MGB; The 5′ end of the probe for detecting Salmonella is connected to fluorescein FAM, and the 3′ end is connected to fluorescein BHQ1; The 5' end of the probe for detecting Staphylococcus aureus is connected to fluorescein CY5, and the 3' end is connected to fluorescein MGB.

2. Use of the primer pair and probe set according to claim 1 in the preparation of a multiplex digital PCR detection product for detecting Campylobacter, Salmonella and Staphylococcus aureus.

Images