CN116064957A - Multiplex real-time fluorescent PCR detection kit for detecting viral pathogen causing bovine diarrhea and application thereof - Google Patents
Multiplex real-time fluorescent PCR detection kit for detecting viral pathogen causing bovine diarrhea and application thereof Download PDFInfo
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Abstract
The invention discloses a multiplex real-time fluorescent PCR detection kit for detecting viral pathogens causing bovine diarrhea and application thereof. The virus pathogen causing bovine diarrhea disease comprises bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, and the multiplex real-time fluorescent PCR detection kit contains a real-time fluorescent PCR primer pair and SYBRGreen dye which are respectively used for detecting the viruses. The invention designs specific primers aiming at the highly conserved regions of the 4 virus target genes respectively through gene sequence comparison, and establishes a multiplex SYBRGreen real-time fluorescence PCR method for simultaneously detecting 4 viruses through reaction condition optimization. The detection sensitivity of the method is obviously higher than that of a conventional PCR method, and meanwhile, the method has good detection specificity and repeatability, and provides a technical means for rapid differential diagnosis of BVDV, BCoV, BRV and BPV.
Description
Technical Field
The invention relates to a multiplex real-time fluorescent PCR detection kit for detecting viral pathogens causing bovine diarrhea and application thereof, in particular to a multiplex real-time fluorescent PCR detection kit for detecting Bovine Viral Diarrhea Virus (BVDV), bovine Rotavirus (BRV), bovine coronavirus (BCoV) and Bovine Parvovirus (BPV) and application thereof. The invention belongs to the field of biotechnology.
Background
Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV), bovine coronavirus (Bovine Coronavirus, BCoV), bovine Rotavirus (BRV) and Bovine parvovirus (Bovine parvovirus, BPV) are the main viral pathogens causing Bovine diarrhea, and all clinical symptoms are characterized by diarrhea, and are often mixed infections, which are difficult to identify clinically. Currently, a multiplex real-time fluorescent PCR method for detecting BVDV, BRV, BPV, BVDV+BPV+BPIV, BVDV+IBRV+FMDV, bovine astrovirus+bovine viral diarrhea virus+bovine coronavirus+bovine rotavirus+bovine viral diarrhea virus+infectious bronchitis virus and foot-and-mouth disease virus exists.
Bovine diarrhea caused by BVDV, BCoV, BRV and BPV has similar clinical symptoms, and often causes mixed infection, which is difficult to clinically diagnose. The existing single PCR detection method can achieve the aim of identification detection, but is time-consuming and labor-consuming; in addition, there is no effective method for simultaneously and rapidly identifying and detecting the 4 viruses.
The invention establishes a quadruple real-time fluorescent quantitative PCR method for detecting BVDV, BCoV, BRV and BPV based on the advantage of high flux of the multiplex PCR method and the sensitivity of the fluorescent dye SYBR Green, and assembles a detection kit, thereby providing necessary technical means for clinical rapid differential diagnosis, epidemic situation monitoring and epidemiological investigation.
Disclosure of Invention
The invention aims to provide a multiplex real-time fluorescent PCR detection kit for detecting viral pathogens causing bovine diarrhea and application thereof.
In order to achieve the above purpose, the invention adopts the following technical means:
the invention relates to a multiplex real-time fluorescence PCR detection kit for detecting Bovine diarrhea virus pathogens, wherein the Bovine diarrhea virus pathogens comprise Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV), bovine coronavirus (Bovine Coronavirus, BCoV), bovine Rotavirus (BRV) and Bovine parvovirus (Bovine parvovirus, BPV), and the multiplex real-time fluorescence PCR detection kit is characterized by comprising a real-time fluorescence PCR primer pair and FastStart Universal SYBR Green Master for detecting Bovine diarrhea virus, bovine coronavirus, bovine rotavirus and Bovine parvovirus respectively;
wherein, the real-time fluorescence PCR primer pair for detecting bovine viral diarrhea virus consists of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO. 2;
wherein, the real-time fluorescence PCR primer pair for detecting bovine coronavirus consists of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4;
wherein, the real-time fluorescence PCR primer pair for detecting bovine rotavirus consists of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO. 6;
wherein, the real-time fluorescence PCR primer pair for detecting bovine parvovirus consists of an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO. 8.
Preferably, when the kit is used for detecting bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, the multiplex real-time fluorescence PCR reaction system comprises: fastStart Universal SYBR Green Master 10. Mu.L, 0.2. Mu.L of each of the upstream and downstream primers for detecting bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, 4. Mu.L of the template, and 20. Mu.L of DEPC treated water.
Preferably, when the kit is used for detecting bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, the multiplex real-time fluorescent PCR reaction conditions are as follows: 95 ℃ for 10min;95℃for 15s and 60℃for 1min.
Furthermore, the invention also provides application of the multiplex real-time fluorescence PCR detection kit in preparing reagents for simultaneously detecting bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus.
Wherein, preferably, the reagent is a real-time fluorescence PCR detection reagent.
Compared with the prior art, the invention has the beneficial effects that:
in order to establish a detection method which is rapid, accurate, high in sensitivity and high in specificity and simultaneously distinguishes BVDV, BCoV, BRV and BPV, the invention respectively selects BVDV5' UTR genes, BCoV NSp10 genes, BRV VP6 genes and BPV VP2 genes as target genes through gene sequence comparison, designs specific primers aiming at highly conserved regions of the target genes, and establishes a multiplex SYBR Green real-time fluorescent PCR method for simultaneously detecting 4 viruses through reaction condition optimization. The specificity test results show that only BVDV, BCoV, BRV and BPV are positive results of the method and other viruses are negative results. The method has the lowest detection limit of BVDV, BCoV, BRV and BPV of 7.77×10 respectively 1 copies/μL、7.11×10 2 copies/μL、1.74×10 2 Copies/. Mu.L and 2.53X10 2 The copies/. Mu.L, and the intra-and inter-group variation coefficients were less than 1.0%, showed good detection specificity and reproducibility. In order to evaluate the practicability of the detection method, 230 cattle clinical diarrhea samples are collected for detection, and meanwhile, a commercial kit is adopted for parallel detection, so that results show that the positive rates of BVDV, BCoV, BRV and BPV are 66.96%, 3.48%, 18.26% and 9.6%, and the coincidence rates with the detection results of the kit are 100%, 99.6%, 100% and 100%, so that good practicability is achieved. Aiming at BVDV, BCoV, BRV and BPV causing bovine diarrhea, specific primers are designed from a molecular angle, and a multiplex SYBRGreen real-time fluorescent quantitative PCR detection method is established. The detection sensitivity of the method is obviously higher than that of a conventional PCR method, and the method has good detection specificity and repeatability. The method and the detection kit have the advantages of convenient use, high detection efficiency, cost saving and detection speedThe advantages of quick sealing detection, no pollution, high sensitivity and the like are provided for the rapid differential diagnosis of BVDV, BCoV, BRV and BPV.
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FIG. 1 is the establishment of a BVDV real-time fluorescent PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BVDV-5' UTR;2: a negative control;
FIG. 2 is the establishment of a BCoV real-time fluorescence RT-PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BCoV-NSp10;2: a negative control;
FIG. 3 is the establishment of BRV real-time fluorescence RT-PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BRV-VP6;2: a negative control;
FIG. 4 is the establishment of a BPV real-time fluorescence PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BPV-VP2;2: a negative control;
FIG. 5 shows the amplification curve (A), the dissolution curve (B) and the standard curve (C) of the real-time fluorescence PCR method for detecting BVDV;
FIG. 6 shows the amplification curve (A), the dissolution curve (B) and the standard curve (C) of BCoV detection by the real-time fluorescence RT-PCR method;
FIG. 7 shows the amplification curve (A), the dissolution curve (B) and the standard curve (C) of BRV detection by the real-time fluorescence RT-PCR method;
FIG. 8 shows the amplification curve (A), the dissolution curve (B) and the standard curve (C) of the detection of BPV by the real-time fluorescence RT-PCR method;
FIG. 9 shows the sensitivity of the real-time fluorescence PCR method for detecting BVDV;
wherein, (A) an amplification curve; (B) dissolution profile; 1-7: plasmid concentration was 7.77×10 6 copies/μL～7.77×10 0 copies/μL；
FIG. 10 shows the sensitivity results of detecting BCoV by a real-time fluorescent RT-PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1-7: plasmid concentration was 7.11X10 × 6 copies/μL～7.11×10 0 copies/μL；
FIG. 11 shows the sensitivity results of real-time fluorescence RT-PCR method for detecting BRV;
wherein, (A) an amplification curve; (B) dissolution profile; 1-8: plasmid concentration was 1.7X10 6 copies/μL～1.74×10 0 copies/μL；
FIG. 12 is a plot of the sensitivity results of the real-time fluorescence PCR method for detecting BPV;
wherein, (A) an amplification curve; (B) dissolution profile; 1-8: plasmid concentration was 2.53X10 6 copies/μL～2.53×10 0 copies/μL；
FIG. 13 shows the specific results of real-time fluorescence PCR method for detecting BVDV;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BVDV-5' UTR;2: BVDV;
FIG. 14 shows the specificity of BcoV detection by real-time fluorescent RT-PCR;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BCoV-NSp10;2: BCoV;
FIG. 15 shows the result of detecting BRV specificity by a real-time fluorescence RT-PCR method;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD19-T-BRV-VP6;2: BRV;
FIG. 16 shows the result of detecting BPV by real-time fluorescence PCR;
wherein, (A) an amplification curve; (B) dissolution profile; 1: pMD-BPV-VP2;2: BPV;
FIG. 17 is the establishment of a multiplex real-time fluorescent PCR method;
FIG. 18 shows the stability of the kit for BVDV detection;
wherein, (A) is stored for 3 months; (B) storing for 6 months; (C) storing for 12 months; 1: cationic control pMD-BVDV-5' UTR;2: BVDV;
FIG. 19 is a stability test result of the kit for detecting BCoV;
wherein, (A) is stored for 3 months; (B) storing for 6 months; (C) storing for 12 months; 1: cationic control pMD-BCoV-NSp10;2: BCoV;
FIG. 20 is a stability test result of the kit for detecting BRV;
wherein, (A) is stored for 3 months; (B) storing for 6 months; (C) storing for 12 months; 1: cationic control pMD-BRV-VP6;2: BRV;
FIG. 21 is a stability test result of the kit for detecting BPV;
wherein, (A) is stored for 3 months; (B) storing for 6 months; (C) storing for 12 months; 1: cationic control pMD-BPV-VP2;2: BPV.
Detailed Description
The invention will be further described with reference to specific examples and figures, which are only intended to illustrate the invention and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1
1. Construction of a Standard substance
By analyzing biological information of BVDV, BCoV, BRV and BPV genomes, the invention determines that BVDV5' -UTR genes, BCoVNSP10 genes, BRVVP6 genes and BPVVP2 genes are target genes to establish a detection method. F1/R1 primer pairs (see Table 1) for amplifying the target genes of viruses are designed, the target genes are amplified and connected into a pMD19-T vector, and the constructed plasmids are named pMD-BVDV-5' UTR, pMD-BCoV-Nsp10, pMD-BRV-VP6 and pMD-BPV-VP2 respectively as standard quality control of a detection method.
TABLE 1 primer sequences for amplifying target genes
By BLAST alignment, the specific primer pair F2/R2 of the detection method was designed for highly conserved regions of the target gene (Table 2). The real-time fluorescence PCR method for detecting BVDV, BCoV, BRV and BPV is established by using the primer pair F2/R2 by using pMD-BVDV-5' UTR, pMD-BCoV-NSp10, pMD-BRV-VP6 and pMD-BPV-VP2 as templates respectively.
TABLE 2 specific primer sequences for the establishment of the detection methods
2. Establishment of single SYBRGreen real-time fluorescence PCR detection method
The single SYBR Green real-time fluorescence PCR method for detecting BVDV (figure 1), BCoV (figure 2), BRV (figure 3) and BPV (figure 4) is established by using standard plasmids pMD-BVDV-5' UTR, pMD-BCoV-Nsp10, pMD-BRV-VP6 and pMD-BPV-VP2 as templates and simultaneously DEPC water control, and using the primer pair F2/R2 for amplification. The reaction system is shown in Table 3. The reaction procedure was 95℃for 10min;95℃for 15s and 60℃for 1min.
TABLE 3 real-time fluorescence PCR method reaction system (20. Mu.L)
3. Drawing of a Standard Curve
At a concentration of 7.77×10 5 copies/μL～7.77×10 9 Real-time fluorescence PCR detection was performed using copies/. Mu.L of plasmid pMD-BVDV-5' UTR as a template, 3 replicates were set for each plasmid concentration, and a standard curve (FIG. 5) for quantitative BVDV detection was established, with the standard curve equation: y= -5.923x+57.703, r 2 =0.998, the linearity is good.
At a concentration of 7.11×10 4 copies/μL～7.11×10 8 Real-time PCR detection was performed using the copies/. Mu.L plasmid pMD-BCoV-NSp10 as a template, 3 replicates were set for each plasmid concentration, and a standard curve (FIG. 6) for quantitative detection of BCoV was established, with the standard curve equation: y= -4.1590+46.169, r 2 =0.999, the linearity is good.
At a concentration of 1.7X10 4 copies/μL～1.74×10 8 The plasmid pMD-BRV-VP6 of copies/. Mu.L is used as a template to carry out real-time fluorescence PCR detection, 3 repeats are set for each concentration, and quantitative detection BR is establishedV standard curve (fig. 7), standard curve equation: y= -4.118x+44.085, r 2 =0.997, the linearity is good.
At a concentration of 2.53X 10 5 copies/μL-2.53×10 9 The copies/. Mu.L plasmid pMD-BPV-VP2 is used as a template for real-time fluorescence PCR detection, 3 repeats are set for each concentration, and a standard curve (FIG. 8) for quantitatively detecting BPV is established, wherein the standard curve equation is as follows: y= -4.067x+48.6, r 2 =0.996, the linearity is good.
4. Detection sensitivity results of the multiplex PCR method
At a concentration of 7.77×10 6 copies/μL～7.77×10 0 The sensitivity was determined using the copies/. Mu.L of plasmid pMD-BVDV-5' UTR as template. As a result, as shown in FIG. 9, the lowest detection limit was 7.7X10 1 copies/μL。
At a concentration of 7.11×10 6 copies/μL～7.11×10 0 The copies/. Mu.L plasmid pMD-BCoV-NSp10 was used as a template for real-time fluorescent PCR detection to determine the detection sensitivity of the method. The results are shown in FIG. 10, and the minimum detection limit of the method is 7.11X10 2 copies/μL。
At a concentration of 1.7 4 ×10 6 copies/μL～1.7 4 ×10 0 The copies/. Mu.L plasmid pMD-BRV-VP6 was used as template for real-time fluorescent PCR detection to determine the detection sensitivity of the method. The results are shown in FIG. 11, and the minimum detection limit of the method is 1.7X10 2 copies/μL。
At a concentration of 2.53X 10 6 copies/μL～2.53×10 0 The sensitivity was detected using the copies/. Mu.L plasmid pMD-BPV-VP2 as a template. As a result, as shown in FIG. 12, the lowest detection limit was 2.53X10 2 copies/μL。
5. Detection specificity results of the multiplex PCR method
The standard plasmid pMD-BVDV-5' UTR is used as positive quality control, the established BVDV real-time fluorescence PCR method is used for detecting BVDV, BCoV, BRV, BPV, BPIV, IBRV, BRSV, TGEV, PCV, VSV and CDV, the result is shown in figure 13, only BVDV has positive amplification curve, the peak value of the dissolution curve is consistent with the positive quality control, other viruses are negative, and good specificity is shown.
The standard plasmid pMD-BCoV-NSp10 is used as positive quality control, the established BCoV real-time fluorescence PCR method is used for detecting BVDV, BCoV, BRV, BPV, BPIV, IBRV, BRSV, TGEV, PCV2, VSV and CDV, the result is shown in FIG. 14, only BCoV has positive amplification curve, the peak value of the dissolution curve is consistent with the positive quality control, other viruses are negative, and good specificity is shown.
The standard plasmid pMD-BRV-VP6 is used as positive quality control, the established BRV real-time fluorescent RT-PCR method is used for detecting BVDV, BCoV, BRV, BPV, BPIV, IBRV, BRSV, TGEV, PCV, VSV and CDV, the result is shown in FIG. 15, only BRV shows positive amplification curve, the peak value of the dissolution curve is consistent with the positive quality control, other viruses are negative, and good specificity is shown.
The standard plasmid pMD-BPV-VP2 is used as positive quality control, the established BPV real-time fluorescence PCR method is used for detecting BVDV, BCoV, BRV, BPV, BPIV, IBRV, BRSV, TGEV, PCV, VSV and CDV, the result is shown in FIG. 16, only the BPV has positive amplification curve, the peak value of the dissolution curve is consistent with the positive quality control, and other viruses are negative, so that good specificity is shown.
6. Detection reproducibility results of the multiplex PCR method
The concentration of the same batch is 10 respectively by using the established method 6 ～10 8 The concentration of copies/. Mu.L and the different batches was 10 6 ～10 8 Detecting the standard plasmid of cobies/. Mu.L, and calculating the average standard deviation of Ct values
And Coefficient of Variation (CV) to evaluate the detection reproducibility of the method. The results are shown in tables 4, 5, 6 and 7, and the variation coefficients are less than 1.0%, which indicates that the detection repeatability of the method is good.
TABLE 4 repeatability results of the method for detecting BVDV
Table 5 repeatability results of the test BCoV method
Table 6 shows the reproducibility of the BRV method
TABLE 7 repeatability results of the test BPV method
7. Establishment of multiplex SYBRGreen real-time fluorescence PCR detection method
On the basis of the single real-time fluorescence PCR method, a multiple real-time fluorescence PCR reaction system is optimized, a quadruple SYBRGreen real-time fluorescence PCR detection method (figure 17) for simultaneously detecting BVDV, BCoV, BRV, BPV is established, and identification is carried out through corresponding melting curve peak temperatures of 79.96 ℃, 78.72 ℃, 76.99 ℃ and 82.25 ℃. The reaction program of the multiplex real-time fluorescence PCR method is 10min at 95 ℃;95℃for 15s and 60℃for 1min. The reaction system is shown in Table 8.
TABLE 8 quadruple SYBRGreen real-time fluorescence PCR reaction System (20. Mu.L)
8. Detection sensitivity of multiplex real-time fluorescent PCR method
The method is similar to the sensitivity detection of the single real-time fluorescence PCR method, and the detection results are shown in Table 9.
TABLE 9 detection sensitivity of multiplex real-time fluorescent PCR method
9. Detection specificity of multiplex real-time fluorescent PCR method
The method is similar to the specific detection of the single real-time fluorescence PCR method, and the detection results are shown in Table 10.
TABLE 10 detection specificity of multiplex real-time fluorescent PCR method
10. Compliance experiment
In order to verify the accuracy of the detection result of the multiplex real-time fluorescence PCR method, the detection method uses BVDV, BCoV, BRV and BPV commercial nucleic acid detection kits to carry out recheck detection on 230 parts of bovine clinical diarrhea samples, the results are shown in a table 11, the commercial kits detect BVDV, BCoV, BRV and the number of BPV positive samples are 154 parts, 9 parts, 42 parts and 22 parts respectively, the positive rates are 66.96%, 3.91%, 18.26% and 9.57% respectively, and the coincidence rates with the detection method established by the method are 100%, 99.6%, 100% and 100% respectively, so that the detection result of the multiplex real-time fluorescence PCR method established by the method is relatively good in accuracy and can be used for detection of clinical samples.
Table 11 compliance results
11. Stability test of kit
The stability of the assembled kit was evaluated, and the results are shown in fig. 18, 19, 20 and 21, which show that the assembled kit has good stability
12. Kit composition
The kit of the invention comprises: standard plasmids pMD-BVDV-5' UTR, pMD-BCoV-NSp10, pMD-BRV-VP6 and pMD-BPV-VP2; F2/R2 primer pairs; DEPC water; SYBRGreen Master dye.
Claims (5)
1. A multiplex real-time fluorescent PCR assay kit for detecting Bovine diarrhea virus-causing pathogens including Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV), bovine coronavirus (Bovine Coronavirus, BCoV), bovine Rotavirus (BRV) and Bovine parvovirus (Bovine parvovirus, BPV), characterized in that the multiplex real-time fluorescent PCR assay kit comprises a real-time fluorescent PCR primer pair and FastStartUniversal SYBR GreenMaster for detecting Bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and Bovine parvovirus, respectively;
wherein, the real-time fluorescence PCR primer pair for detecting bovine viral diarrhea virus consists of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO. 2;
wherein, the real-time fluorescence PCR primer pair for detecting bovine coronavirus consists of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4;
wherein, the real-time fluorescence PCR primer pair for detecting bovine rotavirus consists of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO. 6;
wherein, the real-time fluorescence PCR primer pair for detecting bovine parvovirus consists of an upstream primer shown in SEQ ID NO.7 and a downstream primer shown in SEQ ID NO. 8.
2. The multiplex real-time fluorescent PCR detection kit according to claim 1, wherein the multiplex real-time fluorescent PCR reaction system comprises: fastStart Universal SYBR Green Master 10. Mu.L, 0.2. Mu.L of each of the upstream and downstream primers for detecting bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, 4. Mu.L of the template, and 20. Mu.L of DEPC treated water.
3. The multiplex real-time fluorescent PCR assay kit as defined in claim 2 wherein, when the kit is used to detect bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus, the multiplex real-time fluorescent PCR reaction conditions are: 95 ℃ for 10min; 15s at 95℃and 1min at 60 ℃.
4. Use of the multiplex real-time fluorescent PCR detection kit as claimed in any one of claims 1 to 3 for the preparation of reagents for simultaneous detection of bovine viral diarrhea virus, bovine coronavirus, bovine rotavirus and bovine parvovirus.
5. The use of claim 4, wherein the reagent is a real-time fluorescent PCR detection reagent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117802275A (en) * | 2024-03-01 | 2024-04-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof |
| CN117802275B (en) * | 2024-03-01 | 2024-05-17 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof |
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2022
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117802275A (en) * | 2024-03-01 | 2024-04-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof |
| CN117802275B (en) * | 2024-03-01 | 2024-05-17 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof |
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