CN114657273A - Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application thereof - Google Patents
Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pair and a probe combination for detecting various bovine mastitis pathogens, wherein the primer pair and the probe respectively aim at staphylococcus aureus rpoB gene, streptococcus tuf gene, mycoplasma bovis oppD/F gene, staphylococcus chromogenes sodA gene and escherichia coli 79 gene, and the sequences are shown as SEQ ID NO. 1-15. The primer pair and the probe combination disclosed by the invention can be used for simultaneously detecting staphylococcus aureus, streptococcus (streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis), mycoplasma bovis, staphylococcus chromogenes and escherichia coli in clinical milk samples or other samples, so that the detection efficiency can be improved, and the sensitivity and the specificity are higher.
Description
Technical Field
The invention belongs to the field of pathogen detection, and relates to a primer pair and probe combination for detecting bovine mastitis pathogens, application of the primer pair and probe combination, and a TaqMan real-time fluorescence quantitative PCR method for detecting various bovine mastitis pathogens in a non-diagnosis purpose.
Background
Bovine mastitis is one of the most important diseases for restricting the development of the dairy cow breeding industry all over the world, not only causes huge economic loss, including milk yield reduction, milk quality reduction and additional treatment and disposal cost, but also causes serious threat to public health safety threat due to antibiotic abuse and multi-drug resistant bacteria during treatment. Worldwide, the incidence of clinical mastitis in cows is about 30% and the prevalence of subclinical mastitis is 15% -75% (Molineri et al, Prev Vet Med,2021,188: 105261). The types of pathogenic bacteria causing bovine mastitis are many, and in China, staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli cause most of bovine mastitis (Song X et al, Veterinary Microbiology 2020,247: 108757; Liu Y et al, Prev Vet Med 2020,182: 105106). Accurately identifies bovine mastitis pathogenic bacteria, and is favorable for early discovery and timely treatment.
At present, the incidence rate of mastitis of domestic dairy cows is high, and an accurate and efficient diagnosis method is lacked. The commonly used microbial culture method is considered as the 'gold standard', but has the disadvantages of long time consumption (5-7 days), low accuracy and high requirement on operators; serological detection methods such as ELISA, immune colloidal gold technology and the like have diagnosis 'window period', and a certain period is needed for an animal body to generate a corresponding detection target; ordinary PCR is most widely used, but electrophoresis and sequencing are required after amplification to obtain results.
Because a single-tube multi-pathogen one-time detection method is lacked in the field of cow mastitis diagnosis, the fluorescence quantitative PCR is widely used for animal pathogen detection, and multi-pathogen and result visual detection can be realized based on the technology. Therefore, the research combines the clinical detection requirements with the method, establishes a TaqMan fluorescent quantitative PCR method capable of detecting seven main pathogens of the bovine mastitis, fills the blank of the detection and clinical application of the mastitis pathogen of the dairy cows in a laboratory, and has great economic benefit and social benefit.
Disclosure of Invention
The primer pair and the probe combination provided by the invention can be used for simultaneously detecting staphylococcus aureus, streptococcus (streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis), mycoplasma bovis, staphylococcus chromogenes and escherichia coli in clinical milk samples or other samples, so that the detection efficiency can be improved, and the primer pair and the probe combination have higher sensitivity and specificity.
The primer pair and the probe combination provided by the invention are respectively designed aiming at staphylococcus aureus rpoB gene, streptococcus tuf gene, mycoplasma bovis oppD/F gene, staphylococcus chromogenes sodA gene and escherichia coli No. 79 gene, wherein,
the sequence of a primer pair aiming at the rpoB gene of staphylococcus aureus is shown as SEQ ID NO 1-2, and the sequence of a probe is shown as SEQ ID NO 3;
the primer pair sequence aiming at the streptococcal tuf gene is shown as SEQ ID NO. 4-5, and the probe sequence is shown as SEQ ID NO. 6;
the primer pair sequence aiming at the OPpD/F gene of the mycoplasma bovis is shown as SEQ ID NO. 7-8, and the probe sequence is shown as SEQ ID NO. 9;
the primer pair sequence aiming at the staphylococcus chromogenes sodA gene is shown as SEQ ID NO 10-11, and the probe sequence is shown as SEQ ID NO 12;
the sequence of the primer pair aiming at the No. 79 gene of the escherichia coli is shown as SEQ ID NO. 13-14, and the sequence of the probe is shown as SEQ ID NO. 15.
Wherein, the 5 'end of the probe is marked with a report group, and the 3' end of the probe is marked with a quenching group.
Preferably, the 5 '-end labeled reporter group of the probe for detecting staphylococcus aureus is FAM, and the 3' -end labeled quenching group is BHQ 1; the 5 '-end labeled reporter group of the probe for detecting streptococcus is VIC, and the 3' -end labeled quenching group is BHQ 1; the 5 '-end labeled reporter group of the probe for detecting the mycoplasma bovis is CY5, and the 3' -end labeled quenching group is BHQ 2; the 5 '-end labeled reporter group of the probe for detecting the staphylococcus chromogenes is Texas red, and the 3' -end labeled quenching group is BHQ 2; the 5 'labeled reporter group of the probe for detecting E.coli was CY5.5, and the 3' labeled quencher group was BHQ 3.
Another object of the invention is to provide the use of the primer pair and probe combination in the detection of various bovine mastitis pathogens, such as Staphylococcus aureus, Streptococcus, Mycoplasma bovis, Staphylococcus chromogenes, and Escherichia coli. The application can be used for disease diagnosis of bovine mastitis, and can also be used for laboratory screening and identification of bovine mastitis pathogens for non-diagnosis purposes, food safety inspection and the like.
Wherein the streptococcus is streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis.
The third purpose of the invention is to provide a kit for detecting a plurality of bovine mastitis pathogens, which comprises the primer pair and the probe combination.
The fourth purpose of the invention is to provide a TaqMan real-time fluorescence quantitative PCR method for detecting various bovine mastitis pathogens with non-diagnostic purpose, which comprises the step of taking a detection sample as a template, adding reaction liquid, ultrapure water and the primer pair and the probe set which are combined for PCR amplification.
Preferably, the PCR amplification system is: 2 XProbe PCR Master Mix 10 uL; the final concentrations of the primers on the upstream and downstream of the staphylococcus aureus are both 450nmol/L, and the final concentration of the probe is 175 nmol/L; upstream and downstream of streptococcusThe final concentration of the primers is 450nmol/L, and the final concentration of the probe is 125 nmol/L; the final concentration of the upstream and downstream primers of the mycoplasma bovis is 400nmol/L, and the final concentration of the probe is 175 nmol/L; the final concentrations of the upstream primer and the downstream primer of the staphylococcus chromogenes are both 400nmol/L, and the final concentration of the probe is 125 nmol/L; the final concentrations of the upstream and downstream primers of the escherichia coli are both 400nnol/L, and the final concentration of the probe is 200 nmol/L; template 5. mu.L, ddH2Make up to 20. mu.L of O.
Preferably, the PCR amplification procedure is, 37 ℃ contamination digestion 2 min; pre-denaturation at 95 ℃ for 30s for 1 cycle; annealing at 95 ℃ for 10s and 56.7 ℃ for 30s, and 40 cycles.
The invention has the beneficial effects that:
(1) convenience is provided. The invention can detect 7 pathogens of bovine mastitis at one time by a single tube without respectively designing primers for PCR amplification, can realize the qualitative and quantitative determination of the pathogens according to the Ct value after reaction, and does not need to perform electrophoresis detection on an amplification product, thereby greatly improving the detection efficiency.
(2) And (4) sensitivity. The detection sensitivity of the method established by the invention to the recombinant plasmids is respectively staphylococcus aureus 101copies/. mu.L, Streptococcus 101copies/. mu.L, M.bovis 102copies/. mu.L, Staphylococcus chromogenes 101copies/. mu.L, E.coli 101copies/. mu.L. Compared with the common PCR method, the method established by the invention has higher detection sensitivity.
(3) Specificity. When the method is used for qPCR amplification, a specific fluorescence curve can be obtained for staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli, while positive nucleic acids of staphylococcus epidermidis, mycoplasma agalactiae, klebsiella pneumoniae, bacillus licheniformis, cryptococcus pyogenes, bacillus curvatus, bacillus cereus and lactococcus garvieae cannot generate amplification curves and have no cross reactivity with other bacteria.
Drawings
FIG. 1 shows the sensitivity of the method of the present invention to Staphylococcus aureus.
FIG. 2 shows the susceptibility of the method of the present invention to Streptococcus.
FIG. 3 is the sensitivity of the method established by the present invention to M.bovis.
FIG. 4 is a graph showing the sensitivity of the method of the present invention to Staphylococcus chromogenes.
FIG. 5 shows the sensitivity of the method of the present invention to E.coli.
FIG. 6 shows the sensitivity of the general PCR method to the detection of five bovine mastitis pathogens, wherein A is Staphylococcus aureus, B is streptococcus, C is Mycoplasma bovis, D is Staphylococcus chromogenes, E is Escherichia coli, M is molecular weight, 1-7 are respectively 1 × 105copies/μL-1×100copies/. mu.L total 6 gradients and blank.
FIG. 7 shows the specificity of detection according to the invention.
Detailed Description
The present invention will be described in detail below with reference to specific examples.
Example 1 primer sequences and optimization
Primers and probes were designed based on NCBI published rpoB gene of Staphylococcus aureus (YP _499096.2), tuf gene of Streptococcus agalactiae (NZ _ AP018400.1), oppD/F gene of Mycoplasma bovis (AF130119.1), sodA gene of Staphylococcus chromogenes (AJ343945.1) and gene sequence No. 79 of Escherichia coli (NZ _ JADEVB 010000078.1). In order to improve the specificity of designing a probe based on the rpoB gene, the 4 th base of an upstream probe is changed into a degenerate base Y; in order to realize the detection of three main streptococcus including streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis, tuf gene sequences of the three streptococcus are determined, probes and primers are optimized according to sequencing sequences, the 14 th base, the 16 th base and the 20 th base of an upstream probe are respectively changed into the degenerate bases R, Y and W, and the 15 th base of a downstream primer is changed into R. The sequences of the primers and probes are shown in Table 1, and the sequence of the target fragment is shown in Table 2. The primers and probes were synthesized by Biotech, Inc. of Ongbenaceae, Beijing, using ddH2Diluting to a concentration of 10 mu mol/L by using O, and storing at 4 ℃ for later use.
TABLE 1 primer and Probe sequences
TABLE 2 sequences of fragments of interest
Example 2 optimization of assay conditions
At a concentration of 1X 106The recombinant plasmids of the copes/mu L staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli are used as templates, a 20 mu L reaction system and 2 XProbe PCR Master Mix 10 mu L are adopted, the final concentration of the upstream primer, the downstream primer and the Probe primer is adjusted between 100nmol/L and 500nmol/L, and the template is 5 mu L (1 mu L of each of 5 recombinant plasmids) ddH2Make up to 20. mu.L of O. The reaction procedure is as follows: digesting for 2min at 37 deg.C; pre-denaturation at 95 ℃ for 30s for 1 cycle; annealing at 95 deg.C for 10s and 50-65 deg.C for 30s, and repeating for 40 cycles. And determining the optimal primer probe concentration and annealing temperature according to the fluorescent quantitative PCR amplification curve and the Ct value.
The optimized reaction conditions are as follows: 2 XProbe PCR Master Mix 10 uL, the final concentration of the staphylococcus aureus upstream and downstream primers is 450 nmol/L; the final concentration of the streptococcus upstream and downstream primers is 450 nmol/L; the final concentration of the upstream and downstream primers of the mycoplasma bovis is 400 nmol/L; the final concentrations of the upstream primer and the downstream primer of the staphylococcus chromogenes are both 400 nmol/L; the final concentration of the primers on the upstream and downstream of the Escherichia coli is 400 nnol/L. The final concentration of the staphylococcus aureus probe is 175 nmol/L; the final concentration of the streptococcus probe is 125 nmol/L; the final concentration of the mycoplasma bovis probe is 175 nmol/L; the final concentration of the staphylococcus chromogenes probe is 125 nmol/L; the final concentration of the escherichia coli probe is 200 nmol/L; template 5. mu.L, ddH2Make up to 20 μ L O, the reaction sequence was: digesting the pollution for 2min at 37 ℃; pre-denaturation at 95 ℃ for 30s for 1 cycle; 10s at 95 ℃, 30s at 56.7 ℃ and 40 cycles.
Example 3 Standard Curve, sensitivity, specificity, reproducibility of the detection method
(1) Standard curve: at 1 × 108copies/μL-1×104The method comprises the steps of taking copies/mu L of 5 gradient-concentration recombinant plasmids of staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli as templates (1 mu L of each 5 recombinant plasmids with the same concentration are added into the same system, and 5 mu L of the recombinant plasmids are added in total), carrying out amplification according to optimized quintuple TaqMan real-time fluorescence quantitative PCR reaction conditions, and establishing a standard curve.
The method takes the lg (x) value of the copy number of the plasmid as an abscissa and the Ct value as an ordinate to establish a standard curve, and the standard curve of the staphylococcus aureus is as follows: -3.358x +40.003, R20.998, and the amplification efficiency E98.5%; streptococcus standard curve: -3.309x +41.426, R20.998, and the amplification efficiency E is 100.6%; staphylococcus chromogenes standard curve y ═ 3.410x +40.713, R20.998, amplification efficiency E96.5%; the standard curve of mycoplasma bovis is: -3.322x +42.031, R20.998, and the amplification efficiency E is 100%; the standard curve of E.coli is: -3.473x +41.651, R2The amplification efficiency E was 94% at 0.998.
(2) Sensitivity: the sensitivity of the method established by the invention is compared with the sensitivity of the common PCR method, and the comparison method comprises the following steps: at 1 × 105copies/μL-1×100The method comprises the steps of taking 6 gradient recombinant plasmids of staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli as templates and double distilled water as negative control templates, carrying out amplification according to optimized quintuple TaqMan real-time fluorescence quantitative PCR reaction conditions, and comparing with a common PCR amplification result by using primers in the method.
The invention has higher sensitivity, and the lowest copy number capable of being detected is staphylococcus aureus 101copies/. mu.L, Streptococcus 101copies/. mu.L, Mycoplasma bovis 102copies/. mu.L, Staphylococcus chromogenes 101copies/. mu.L, E.coli 101copies/. mu.L, sensitivity test results are shown in FIGS. 1-5. The lowest copy number capable of being detected by the common PCR method is staphylococcus aureus 103copies/. mu.L, Streptococcus 104copies/. mu.L, Mycoplasma bovis 103copies/. mu.L, Staphylococcus chromogenes 103copies/. mu.L, E.coli 103copies/. mu.L, sensitivity test results are shown in FIG. 6.
(3) Specificity: when staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli genome DNA are added into a reaction system as templates, and the method established by the application is adopted to carry out real-time fluorescence quantitative PCR amplification, the result obtains a specific fluorescence curve. Amplification results of positive nucleic acids of staphylococcus epidermidis, mycoplasma agalactiae, klebsiella pneumoniae, bacillus licheniformis, cryptococcus pyogenes, bacillus flexus, bacillus cereus and lactococcus garvieae show that DNA, negative control and blank control of the 8 bacteria cannot generate amplification curves and show negative results, and the result shown in figure 7 shows that the real-time fluorescence quantitative PCR method established by the application has good specificity. In FIG. 7, 1 is Staphylococcus aureus; 2-1 is streptococcus agalactiae; 2-2 is streptococcus dysgalactiae; 2-3 is Streptococcus uberis; 3 is mycoplasma bovis; 4 is staphylococcus chromogenes; 5 is Escherichia coli; 6 is staphylococcus epidermidis; 7 is a milk-free mycoplasma; 8 is Klebsiella pneumoniae; 9 is bacillus licheniformis; 10 is Cryptobacterium pyogenes; 11 is campylobacter; 12 is bacillus cereus; 13 is lactococcus garvieae; blank control 14.
(4) Repeatability: respectively at a concentration of 1 × 108copies/μL-1×105The recombinant plasmids of the copes/. mu.L staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli are taken as templates and are divided into 4 samples for simultaneous detection. The template stored at-20 ℃ is repeatedly detected on the 4 th day and the 8 th day, the intra-group repeatability and the inter-group repeatability of the method are verified by calculating the mean value +/-standard deviation (mean +/-SD) and the Coefficient of Variation (CV) of the Ct value, the repeatability test result shows that the intra-group coefficient of variation and the inter-group coefficient of variation of the method are both less than 3 percent, and the detailed data are shown in Table 3.
TABLE 3 repeatability tests
Example 4 clinical testing of bovine mastitis pathogen
Clinical samples were collected from the dairy farms by the following method: 1. marking a centrifugal tube for sampling, wherein the centrifugal tube comprises a date, a cow number, a breast area and a serial number; 2. cleaning the dairy cow breast area and squeezing out the first three milks; 3. disinfecting the nipples, and drying the nipples by using a cleaning paper towel; 4. wiping the nipple with alcohol cotton ball, and collecting milk sample after drying. The method is used for detecting 40 collected clinical milk samples, and comprises the following specific operations: and (3) turning the centrifuge tube filled with the milk sample upside down and uniformly mixing, taking 5 mu L of milk sample, directly adding into the prepared reaction system, putting into a CFX 96 fluorescent quantitative PCR instrument, and judging the bacterial species in the milk sample according to an amplification curve and a Ct value after amplification is finished. Simultaneously, detecting by bacteria separation and identification method, spreading 100 μ L of the collected clinical sample in stock solution, diluting 10 times, 100 times and 1000 times respectively, adding into brain heart infusion solid culture medium, placing at 37 deg.C and 5% CO2Culturing for 24h in an incubator, selecting a single colony for purification according to the shape and size of the colony on each flat plate, selecting the single colony for gram staining after twice purification, transferring a liquid culture medium for culture for 8h-12h after determining that the shape and the size are all consistent, extracting bacterial DNA after the culture medium is turbid, then performing bacterial 16S rDNA amplification and sequencing identification, and performing sequence comparison by using a BLAST database to identify the bacterial species. The detection result of the method is compared with a bacteria separation and identification method, the result shows that the coincidence rate is 100 percent, and the method is proved to be applicable to clinical detection. The detailed results are shown in Table 4.
TABLE 4 test results on clinical specimens
Note: the detection method has the following judgment standards: the Ct value is less than 35, the result is positive, the Ct value is less than 35, the result is suspicious, and the Ct value is more than or equal to 40, the result is negative.
In conclusion, the real-time fluorescent quantitative PCR detection method established by the application has high sensitivity and good specificity, and has no cross reaction with other bacteria; the kit is suitable for routine detection of seven main pathogens of bovine mastitis in laboratories, and can provide necessary technical support for rapid diagnosis of various diseases caused by infection of staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli.
<110> university of agriculture in Huazhong
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Claims (9)
1. A primer pair and probe combination for detecting a plurality of bovine mastitis pathogens is characterized in that: the plurality of bovine mastitis pathogens are staphylococcus aureus, streptococcus, mycoplasma bovis, staphylococcus chromogenes and escherichia coli, the primer pair and the probe respectively aim at staphylococcus aureus rpoB gene, streptococcus tuf gene, mycoplasma bovis oppD/F gene, staphylococcus chromogenes sodA gene and escherichia coli 79 gene, wherein,
the sequence of a primer pair aiming at the rpoB gene of staphylococcus aureus is shown as SEQ ID NO 1-2, and the sequence of a probe is shown as SEQ ID NO 3;
the primer pair sequence aiming at the streptococcal tuf gene is shown as SEQ ID NO. 4-5, and the probe sequence is shown as SEQ ID NO. 6;
the primer pair sequence aiming at the OPpD/F gene of the mycoplasma bovis is shown as SEQ ID NO. 7-8, and the probe sequence is shown as SEQ ID NO. 9;
the primer pair sequence aiming at the staphylococcus chromogenes sodA gene is shown as SEQ ID NO 10-11, and the probe sequence is shown as SEQ ID NO 12;
the sequence of the primer pair aiming at the No. 79 gene of the escherichia coli is shown as SEQ ID NO. 13-14, and the sequence of the probe is shown as SEQ ID NO. 15.
2. The primer pair and probe combination of claim 1, wherein: the 5 'end of the probe is marked with a reporter group, and the 3' end of the probe is marked with a quenching group.
3. The primer pair and probe combination of claim 2, wherein: the 5 'end of the probe aiming at the rpoB gene of the staphylococcus aureus is marked with a reporter group FAM, and the 3' end of the probe is marked with a quenching group BHQ 1;
the 5 '-labeled reporter group of the probe for the streptococcal tuf gene is VIC, and the 3' -labeled quencher group is BHQ 1;
the 5 'end marked reporter group of the probe aiming at the OPpD/F gene of the mycoplasma bovis is CY5, and the 3' end marked quenching group is BHQ 2;
the 5 '-end labeled reporter group of the probe for the staphylococcus chromogenes sodA gene is Texas red, and the 3' -end labeled quenching group is BHQ 2;
the 5 '-labeled reporter group of the probe for the E.coli gene No. 79 was CY5.5, and the 3' -labeled quencher group was BHQ 3.
4. Use of a primer pair and probe combination according to any one of claims 1-3 for the detection of a plurality of bovine mastitis pathogens, which are staphylococcus aureus, streptococcus, mycoplasma bovis, staphylococcus chromogenes, escherichia coli, for non-diagnostic purposes.
5. The use of claim 4, wherein: the streptococcus is Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis.
6. The utility model provides a detect kit of multiple bovine mastitis pathogen which characterized in that: the kit comprises a primer pair and a probe combination according to any one of claims 1 to 3.
7. A TaqMan real-time fluorescent quantitative PCR method for non-diagnosis purpose detection of various bovine mastitis pathogens is characterized in that: the method comprises the step of performing PCR amplification by using a detection sample as a template and adding a reaction solution, ultrapure water and the primer pair and probe set of any one of claims 1 to 3.
8. The TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens according to claim 7, wherein the PCR amplification system comprises: 2 × Probe PCR Master Mix 10 μ L; the final concentrations of the upstream primer and the downstream primer aiming at the rpoB gene of staphylococcus aureus are both 450nmol/L, and the final concentration of the probe is 175 nmol/L; the final concentrations of the upstream primer and the downstream primer aiming at the streptococcal tuf gene are both 450nmol/L and the final concentration of the probe is 125 nmol/L; the final concentrations of the upstream primer and the downstream primer aiming at the OPpD/F gene of the mycoplasma bovis are both 400nmol/L, and the final concentration of the probe is 175 nmol/L; the final concentrations of the upstream primer and the downstream primer aiming at the chromogenes staphylococcus sodA gene are both 400nmol/L, and the final concentration of the probe is 125 nmol/L; the final concentrations of the upstream primer and the downstream primer aiming at the No. 79 gene of the escherichia coli are both 400nnol/L, and the final concentration of the probe is 200 nmol/L; template 5. mu.L, ddH2Make up to 20. mu.L of O.
9. The TaqMan real-time fluorescent quantitative PCR method for the detection of a plurality of bovine mastitis pathogens of claim 7, wherein the PCR amplification procedure is a contaminated digestion at 37 ℃ for 2 min; pre-denaturation at 95 ℃ for 30s for 1 cycle; annealing at 95 ℃ for 10s and 56.7 ℃ for 30s, and 40 cycles.
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| CN105420379A (en) * | 2015-12-24 | 2016-03-23 | 金宇保灵生物药品有限公司 | Real-time fluorescence quantification PCR detecting kit for cow mycoplasma and special primers and TaqMan probe thereof |
| CN108265120A (en) * | 2018-01-09 | 2018-07-10 | 深圳市易瑞生物技术股份有限公司 | A kind of 16 garget pathogenic bacteria nucleic acid parting kits and its detection method |
| CN110938704A (en) * | 2019-12-31 | 2020-03-31 | 吉林工商学院 | LAMP (loop-mediated isothermal amplification) synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk |
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| CN105420379A (en) * | 2015-12-24 | 2016-03-23 | 金宇保灵生物药品有限公司 | Real-time fluorescence quantification PCR detecting kit for cow mycoplasma and special primers and TaqMan probe thereof |
| CN108265120A (en) * | 2018-01-09 | 2018-07-10 | 深圳市易瑞生物技术股份有限公司 | A kind of 16 garget pathogenic bacteria nucleic acid parting kits and its detection method |
| CN110938704A (en) * | 2019-12-31 | 2020-03-31 | 吉林工商学院 | LAMP (loop-mediated isothermal amplification) synchronous detection method and kit for staphylococcus aureus and salmonella in liquid milk |
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