CN114657273B - Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application of primer pair and probe combination - Google Patents
Primer pair and probe combination for detecting multiple bovine mastitis pathogens and application of primer pair and probe combination Download PDFInfo
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Abstract
The application discloses a primer pair and probe combination for detecting multiple bovine mastitis pathogens, wherein the primer pair and the probe are respectively aimed at staphylococcus aureus rpoB gene, streptococcus tuf gene, mycoplasma bovis oppD/F gene, staphylococcus chromogenes sodA gene and escherichia coli No. 79 gene, and the sequences are shown as SEQ ID NO. 1-15. The primer pair and the probe combination disclosed by the application can be used for simultaneously detecting staphylococcus aureus, streptococcus (streptococcus agalactiae, streptococcus agalactiae 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 application 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 a plurality of bovine mastitis pathogens by non-diagnostic purposes.
Background
Bovine mastitis is one of the most important diseases restricting the development of the global dairy cow breeding industry, and not only causes huge economic losses, including milk yield reduction, milk quality reduction and additional treatment and treatment costs, but also causes serious threat to public health safety threat caused by antibiotic abuse and multi-drug resistant bacteria during treatment. On a global scale, the incidence of clinical mastitis in cows is about 30% and the incidence of subclinical mastitis is 15% -75% (molini et al, prev Vet Med,2021, 188:105261). The pathogenic species responsible for bovine mastitis are numerous and in our country staphylococcus aureus, streptococcus agalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes, escherichia coli, which cause most bovine mastitis (Song X et al Veterinary Microbiology,2020,247:108757;Liu Y et al., prev Vet Med 2020, 182:105106). The method can accurately identify the bovine mastitis pathogenic bacteria, and is favorable for early detection and timely treatment.
At present, the incidence rate of cow mastitis in China is high, and an accurate and efficient diagnosis method is lacking. The commonly used microorganism culture method is considered as a 'gold standard', but has the defects of long time consumption (5-7 days), low accuracy and high requirements on operators; serological detection methods such as ELISA, immune colloidal gold technology and the like have a diagnosis 'window period', and a certain period is needed for an animal organism to generate a corresponding detection target; common PCR is most widely used, but electrophoresis and sequencing are required after amplification to obtain the result.
Because of the lack of a single-tube multiple pathogen disposable detection method in the dairy cow mastitis diagnosis field, the fluorescent quantitative PCR is widely used for detecting animal pathogens, and the technology can realize multiple pathogen and result visual detection. Therefore, the research combines the clinical requirement and the method, establishes a TaqMan fluorescent quantitative PCR method capable of detecting seven main pathogens of bovine mastitis, fills the blank of detection and clinical application of the dairy cow mastitis pathogens in a laboratory, and has great economic and social benefits.
Disclosure of Invention
The application provides a primer pair and a probe combination for detecting multiple bovine mastitis pathogens, which can detect staphylococcus aureus, streptococcus (streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis), mycoplasma bovis, staphylococcus chromogenes and escherichia coli in clinical milk samples or other samples at the same time, and can improve the detection efficiency, and have higher sensitivity and specificity.
The primer pair and the probe combination provided by the application are respectively designed for staphylococcus aureus rpoB gene, streptococcus tuf gene, mycoplasma bovis oppD/F gene, staphylococcus chromogenes sodA gene and escherichia coli 79 gene, wherein,
the primer pair sequence for the staphylococcus aureus rpoB gene is shown as SEQ ID NO. 1-2, and the probe sequence is shown as SEQ ID NO. 3;
the primer pair sequence for the tuf gene of streptococcus is shown as SEQ ID NO. 4-5, and the probe sequence is shown as SEQ ID NO. 6;
the primer pair sequence for mycoplasma bovis oppD/F gene is shown in SEQ ID NO. 7-8, and the probe sequence is shown in SEQ ID NO. 9;
the primer pair sequence for 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 primer pair sequence for the No. 79 gene of the escherichia coli is shown as SEQ ID NO. 13-14, and the probe sequence is shown as SEQ ID NO. 15.
Wherein the 5 'end of the probe is marked with a reporter group, and the 3' end is marked with a quenching group.
Preferably, the reporter group labeled at the 5 'end of the probe for detecting staphylococcus aureus is FAM and the quencher group labeled at the 3' end is BHQ1; the reporter group marked at the 5 'end of the probe for detecting streptococcus is VIC, and the quencher group marked at the 3' end is BHQ1; the reporter group marked at the 5 'end of the probe for detecting mycoplasma bovis is CY5, and the quenching group marked at the 3' end is BHQ2; the reporter group marked at the 5 'end of the probe for detecting staphylococcus chromogenes is Texas red, and the quenching group marked at the 3' end is BHQ2; the reporter group labeled at the 5 'end of the probe for detecting E.coli is CY5.5 and the quencher group labeled at the 3' end is BHQ3.
It is another object of the present application to provide the use of the primer pair and probe combination for detecting a plurality of bovine mastitis pathogens, the plurality of bovine mastitis pathogens being staphylococcus aureus, streptococcus, mycoplasma bovis, staphylococcus chromogenes, escherichia coli. The application not only can carry out disease diagnosis on bovine mastitis, but also can be used for screening and identifying bovine mastitis pathogen laboratory for non-diagnosis purposes, food safety inspection and the like.
Wherein the streptococcus is streptococcus agalactiae, streptococcus dysgalactiae and streptococcus uberis.
It is a third object of the present application to provide a kit for detecting a plurality of bovine mastitis pathogens, the kit comprising the primer pair and probe combination described above.
The fourth object of the present application is to provide a TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens without diagnosis, which comprises the steps of taking a detection sample as a template, adding a reaction solution, ultrapure water, and the combination of the primer pair and the probe, and performing PCR amplification.
Preferably, the PCR amplification system is: 2X Probe PCR Master Mix. Mu.L; the final concentration of the upstream and downstream primers of staphylococcus aureus is 450nmol/L, and the final concentration of the probe is 175nmol/L; the final concentration of the primer at the upstream and downstream of the streptococcus is 450nmol/L, and the final concentration of the probe is 125nmol/L; the final concentration of the upstream and downstream primers of mycoplasma bovis is 400nmol/L, and the final concentration of the probe is 175nmol/L; the final concentration of the upstream and downstream primers of the staphylococcus chromogenes is 400nmol/L, and the final concentration of the probe is 125nmol/L; e.coli, the final concentration of the upstream and downstream primers is 400nnol/L, and the final concentration of the probe is 200nmol/L; template 5. Mu.L, ddH 2 O was made up to 20. Mu.L.
Preferably, the PCR amplification procedure is a contamination digestion at 37℃for 2min; pre-denaturation at 95 ℃ for 30s,1 cycle; annealing at 95 ℃ for 10s and 56.7 ℃ for 30s and 40 cycles.
The beneficial effects of the application are as follows:
(1) Convenience. The application can detect 7 pathogens of bovine mastitis at one time by a single tube, does not need to design primers for PCR amplification respectively, can realize the qualitative and quantitative detection of the pathogens according to the Ct value after the reaction, and does not need to carry out electrophoresis detection on the amplified products, thereby greatly improving the detection efficiency.
(2) Sensitivity. The detection sensitivity of the method established by the application to the recombinant plasmid is staphylococcus aureus 10 respectively 1 COPIES/. Mu.L, streptococcus 10 1 COPIES/. Mu.L, mycoplasma bovis 10 2 copies/. Mu.L, staphylococcus chromogenes 10 1 COPIES/. Mu.L, E.coli 10 1 COPies/. Mu.L. Compared with the common PCR method, the method provided by the application has higher detection sensitivity.
(3) Specificity. When qPCR amplification is carried out by the method, specific fluorescence curves can be obtained for staphylococcus aureus, streptococcus agalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli, and amplification curves can not be generated for positive nucleic acids of staphylococcus epidermidis, mycoplasma agalactiae, klebsiella pneumoniae, bacillus licheniformis, cryptobacter suppuration, bacillus curvatus, bacillus cereus and lactococcus grignard, and cross reactivity with other bacteria is avoided.
Drawings
FIG. 1 is a graph showing the sensitivity of the established method of the application to Staphylococcus aureus.
FIG. 2 is a graph showing the sensitivity of the established method of the application to Streptococci.
FIG. 3 is a graph showing the sensitivity of the established method of the application to Mycoplasma bovis.
FIG. 4 is a graph showing the sensitivity of the established method of the application to Staphylococcus chromogenes.
FIG. 5 shows the sensitivity of the established method of the application to E.coli.
FIG. 6 shows the sensitivity of a conventional PCR method to 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, and 1-7 are 1×10 respectively 5 copies/μL-1×10 0 Copies/. Mu.L total 6 gradients and blank.
FIG. 7 is the detection specificity of the present application.
Detailed Description
The present application will be described in detail with reference to the following examples.
Example 1 primer sequences and optimization
Primers and probes were designed based on the rpoB gene of Staphylococcus aureus (YP_ 499096.2), the tuf gene of Streptococcus agalactiae (NZ_AP 018400.1), the oppD/F gene of Mycoplasma bovis (AF 130119.1), the sodA gene of Staphylococcus chromogenes (AJ 343945.1) and the 79-th gene sequence of Escherichia coli (NZ_JADEVB 010000078.1). In order to improve the specificity of the probe designed based on rpoB gene, the 4 th base of the upstream probe is changed into a facultative base Y; in order to realize detection of three main streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis, tuf gene sequences of the three streptococcus agalactiae are determined, probes and primers are optimized according to the sequencing sequences, the 14 th base, the 16 th base and the 20 th base of the upstream probe are respectively changed into a facultative base R, Y and W, and the 15 th base of the downstream primer is changed into R. The sequences of the primers and probes are shown in Table 1, and the sequences of the target fragments are shown in Table 2. Primers and probes were synthesized by Beijing Optimuno Corp., using ddH 2 Diluting O to a concentration of 10 mu mol/L, and storing at 4 ℃ for later use.
TABLE 1 primer and probe sequences
TABLE 2 fragment sequences of interest
Example 2 optimization of detection conditions
At a concentration of 1X 10 6 Recombinant plasmids of staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli with the concentration of the upstream primer and the downstream primer and the probe primer being adjusted between 100nmol/L and 500nmol/L by adopting a 20 mu L reaction system and 2X Probe PCR Master Mix mu L by adopting 20 mu L reaction system as a template, and 5 mu L of the template (1 mu L of each of 5 recombinant plasmids) and ddH 2 O was made up to 20. Mu.L. The reaction procedure is: pollution digestion for 2min at 37 ℃; pre-denaturation at 95 ℃ for 30s,1 cycle; annealing at 95 ℃ for 10s and 50-65 ℃ for 30s and 40 cycles. And determining the optimal primer probe concentration and annealing temperature according to the fluorescence quantitative PCR amplification curve and the Ct value.
The optimized reaction conditions are as follows: 2X Probe PCR Master Mix mu L, and the final concentration of the upstream and downstream primers of staphylococcus aureus is 450nmol/L; the final concentration of the primer at the upstream and downstream of the streptococcus is 450nmol/L; the final concentration of the upstream and downstream primers of mycoplasma bovis is 400nmol/L; the final concentration of the upstream and downstream primers of the staphylococcus chromogenes is 400nmol/L; the final concentration of the upstream and downstream primers of the Escherichia coli is 400nnol/L. The final concentration of the staphylococcus aureus probe is 175nmol/L; the final concentration of the streptococcus probe is 125nmol/L; the final concentration of the mycoplasma bovis probe is 175nmol/L; the final concentration of the chromogenic staphylococcus probe is 125nmol/L; the final concentration of the escherichia coli probe is 200nmol/L; template 5. Mu.L, ddH 2 O was made up to 20. Mu.L, the reaction procedure was: pollution digestion for 2min at 37 ℃; pre-denaturation at 95 ℃ for 30s,1 cycle; annealing at 95 ℃ for 10s and 56.7 ℃ for 30s and 40 cycles.
Example 3 Standard Curve, sensitivity, specificity, repeatability of the detection method
(1) Standard curve: at 1X 10 8 copies/μL-1×10 4 The staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli recombinant plasmids with 5 gradient concentrations are used as templates (1 mu L of 5 recombinant plasmids with the same concentration are added into the same system, and the total amount is 5 mu L), and the amplification is carried out according to optimized five-fold TaqMan real-time fluorescence quantitative PCR reaction conditions, so that a standard curve is established.
The method takes the lg (x) value of the copy number of the plasmid as the abscissa and the Ct value as the ordinate to establish a standard curve, and the method is goldenStandard curve of staphylococcus chromogenes: y= -3.358x+40.003, r 2 =0.998, amplification efficiency e=98.5%; streptococcus standard curve: y= -3.309x+41.426, r 2 =0.998, amplification efficiency e=100.6%; standard curve y = -3.410x +40.713 for staphylococcus chromogenes, r 2 =0.998, amplification efficiency e=96.5%; the mycoplasma bovis standard curve is: y= -3.322x+42.031, r 2 =0.998, amplification efficiency e=100%; the E.coli standard curve is: y= -3.473x+41.651, r 2 =0.998, amplification efficiency e=94%.
(2) Sensitivity: the sensitivity of the method established by the application is compared with that of a common PCR method, and the comparison method is as follows: at 1X 10 5 copies/μL-1×10 0 6 gradient staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli recombinant plasmids are taken as templates, double distilled water is taken as a negative control template, amplification is carried out according to optimized five-fold TaqMan real-time fluorescence quantitative PCR reaction conditions, and the comparison is carried out with the common PCR amplification result by using the primers in the method.
The application has higher sensitivity, and the lowest copy number which can be detected is staphylococcus aureus 10 1 COPIES/. Mu.L, streptococcus 10 1 COPIES/. Mu.L, mycoplasma bovis 10 2 copies/. Mu.L, staphylococcus chromogenes 10 1 COPIES/. Mu.L, E.coli 10 1 The sensitivity test results are shown in FIGS. 1-5. The lowest copy number which can be detected by the common PCR method is staphylococcus aureus 10 3 COPIES/. Mu.L, streptococcus 10 4 COPIES/. Mu.L, mycoplasma bovis 10 3 copies/. Mu.L, staphylococcus chromogenes 10 3 COPIES/. Mu.L, E.coli 10 3 The 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 for real-time fluorescence quantitative PCR amplification, a specific fluorescence curve is obtained as a result. The amplification results of positive nucleic acids of staphylococcus epidermidis, mycoplasma free, klebsiella pneumoniae, bacillus licheniformis, cryptobacillus pyogenes, bacillus curvatus, bacillus cereus and lactococcus garvieae show that the DNA, negative control and blank control of the 8 bacteria can not generate amplification curves, and negative results are shown in figure 7, which 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 agalactiae; 2-3 is streptococcus uberis; 3 is mycoplasma bovis; 4 is staphylococcus chromogenes; 5 is Escherichia coli; 6 is staphylococcus epidermidis; 7 is mycoplasma agalactiae; 8 is Klebsiella pneumoniae; 9 is bacillus licheniformis; 10 is stellera suppurati; 11 is bacillus curvatus; 12 is bacillus cereus; 13 is lactococcus garvieae; 14 is a blank.
(4) Repeatability: at a concentration of 1X 10 respectively 8 copies/μL-1×10 5 The recombinant plasmid of the copies/. Mu.L staphylococcus aureus, streptococcus agalactiae, mycoplasma bovis, staphylococcus chromogenes and escherichia coli is used as a template, and is divided into 4 samples to be detected simultaneously. The templates stored at-20℃were repeatedly examined on days 4 and 8, and the intra-and inter-group repeatability of the method of the present application was verified by calculating the mean.+ -. Standard deviation (mean.+ -. SD) and Coefficient of Variation (CV) of Ct values, and the results of the repeatability test showed that the intra-and inter-group coefficients of variation of the method were less than 3%, and the detailed data are shown in Table 3.
TABLE 3 repeatability test
Example 4 clinical detection of bovine mastitis pathogen
Clinical samples were collected from dairy farms as follows: 1. marking a centrifuge tube for sampling, wherein the centrifuge tube comprises a date, a cow number, a milk area and a number; 2. cleaning a dairy cow milk area and extruding front three milk; 3. sterilizing nippleSucking the nipple with a cleaning paper towel; 4. the nipple is wiped by alcohol cotton ball, and milk sample is collected after the nipple is dried. The method is used for detecting 40 collected clinical milk samples, and the specific operation is as follows: and (3) mixing the centrifuge tube filled with the milk sample upside down, taking 5 mu L of the milk sample, directly adding the milk sample into a prepared reaction system, putting the reaction system into a CFX 96 fluorescent quantitative PCR instrument, and judging the bacterial types in the milk sample according to an amplification curve and a Ct value after amplification. Simultaneously detecting by bacteria separation and identification method, respectively applying 100 μl of collected clinical sample to brain heart infusion solid culture medium at 37deg.C and 5% CO by 10 times, 100 times and 1000 times dilution 2 Culturing in an incubator for 24 hours, selecting single colony for purification according to the form and size of the colony on each flat plate, selecting single colony for gram staining after two times of purification, transferring a liquid culture medium for culturing for 8-12 hours after the form and size are all consistent, extracting bacterial DNA after the culture medium is turbid, then carrying out bacterial 16S rDNA amplification and sequencing identification, and using a BLAST database for sequence comparison identification of bacterial species. The detection result of the method is compared with a bacterial separation identification method, and the result shows that the coincidence rate is 100%, so that the method is proved to be applicable to clinical detection. The detailed results are shown in Table 4.
TABLE 4 detection results on clinical samples
Note that: the detection method has the following judgment criteria: the Ct value is less than 35 and is judged positive, the Ct value is less than 35 and is less than 40 and is judged suspicious, and the Ct value is more than or equal to 40 and is negative.
In conclusion, the real-time fluorescence quantitative PCR detection method established by the application has high sensitivity and good specificity, and has no cross reaction with other bacteria; the method is suitable for routine detection of seven main pathogens of bovine mastitis in a laboratory, and can provide necessary technical support for rapid diagnosis of various diseases caused by staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, mycoplasma bovis, staphylococcus chromogenes and escherichia coli infection.
<110> university of agriculture in China
<120> a primer set and probe set for detecting a plurality of bovine mastitis pathogens and use thereof
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Claims (4)
1. A TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens without diagnosis is characterized by comprising the following steps: the method comprises the steps of taking a detection sample as a template, adding a reaction solution, ultrapure water, a primer pair and a probe, combining and performing PCR amplification, wherein the plurality of bovine mastitis pathogens are staphylococcus aureus, streptococcus, mycoplasma bovis, chromogenic staphylococcus and escherichia coli, the primer pair and the probe are respectively aimed at rpoB genes of the staphylococcus aureus, tuf genes of the streptococcus, oppD/F genes of the mycoplasma bovis, sodA genes of the chromogenic staphylococcus and 79 # genes of the escherichia coli,
the primer pair sequence for the staphylococcus aureus rpoB gene is shown as SEQ ID NO. 1-2, and the probe sequence is shown as SEQ ID NO. 3;
the primer pair sequence for the tuf gene of streptococcus is shown as SEQ ID NO. 4-5, and the probe sequence is shown as SEQ ID NO. 6;
the primer pair sequence for mycoplasma bovis oppD/F gene is shown in SEQ ID NO. 7-8, and the probe sequence is shown in SEQ ID NO. 9;
the primer pair sequence for 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 primer pair sequence for the No. 79 gene of the escherichia coli is shown as SEQ ID NO. 13-14, the probe sequence is shown as SEQ ID NO. 15,
the PCR amplification system comprises: 2X Probe PCR Master Mix. Mu.L; the final concentration of the upstream and downstream primers for the staphylococcus aureus rpoB gene is 450nmol/L, and the final concentration of the probe is 175nmol/L; the final concentration of the upstream and downstream primers for the tuf gene of streptococcus is 450nmol/L, and the final concentration of the probe is 125nmol/L; the final concentration of the upstream and downstream primers for the mycoplasma bovis oppD/F gene is 400nmol/L, and the final concentration of the probe is 175nmol/L; the final concentration of the upstream and downstream primers for the staphylococcus chromogenes sodA gene is 400nmol/L, and the final concentration of the probe is 125nmol/L; the final concentration of the upstream and downstream primers aiming at the No. 79 gene of the escherichia coli is 400nnol/L, and the final concentration of the probe is 200nmol/L; template 5. Mu.L, ddH 2 O is added to 20 mu L;
the PCR amplification procedure is pollution digestion for 2min at 37 ℃; pre-denaturation at 95 ℃ for 30s,1 cycle; annealing at 95 ℃ for 10s and 56.7 ℃ for 30s and 40 cycles.
2. The TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens according to claim 1, wherein: the probe is marked with a report group at the 5 'end and a quenching group at the 3' end.
3. The TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens according to claim 2, wherein: the reporter group marked at the 5 'end of the probe aiming at the staphylococcus aureus rpoB gene is FAM, and the quenching group marked at the 3' end is BHQ1;
the reporter group marked at the 5 'end of the probe for the tuf gene of streptococcus is VIC, and the quencher group marked at the 3' end is BHQ1;
the reporter group marked at the 5 'end of the probe aiming at mycoplasma bovis oppD/F gene is CY5, and the quenching group marked at the 3' end is BHQ2;
the reporter group marked at the 5 'end of the probe aiming at the staphylococcus chromogenes sodA gene is Texas red, and the quenching group marked at the 3' end is BHQ2;
the reporter group labeled at the 5 'end of the probe for E.coli No. 79 gene is CY5.5 and the quencher group labeled at the 3' end is BHQ3.
4. The TaqMan real-time fluorescent quantitative PCR method for detecting a plurality of bovine mastitis pathogens according to claim 1, wherein: the streptococcus is streptococcus agalactiae, streptococcus agalactiae and streptococcus uberis.
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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 |
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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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