CN117802275B - Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof - Google Patents
Triple fluorescence quantitative PCR detection method for main pathogen of calf diarrhea and application thereof Download PDFInfo
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Abstract
The invention discloses a triple fluorescence quantitative PCR detection method for main pathogens of calf diarrhea and application thereof, belonging to the technical field of molecular biology detection. The invention solves the problems of insufficient sensitivity, efficiency, pathogen content measurement and the like of the conventional PCR. The detection method comprises the steps of taking RNA or DNA of a sample to be detected as a template, selecting an N gene of BCoV, an NSP5 gene of BRV and an E.coli K99 gene as detection target genes, comparing target gene sequences, determining a candidate region of a gene sequence to be amplified, designing quantitative PCR amplification primers according to the determined gene sequences, designing probes in the middle of the designed amplification primers, performing fluorescent quantitative PCR detection, and finally analyzing a fluorescent quantitative PCR amplification curve and Ct values to obtain detection results. The detection method, the primer and the probe can be applied to the three virus or bacteria related detection kits, and are applicable to the detection fields of bovine rotavirus, bovine coronavirus and escherichia coli K99.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and relates to a triple fluorescence PCR detection method for bovine rotavirus, bovine coronavirus and escherichia coli K99 and application thereof.
Background
Bovine rotavirus, coronavirus and escherichia coli are important pathogens causing diarrhea disease in cattle. With the large-scale and intensive high development of the dairy cow breeding industry in China, bovine diarrhea, especially newborn calf diarrhea, becomes one of important diseases which afflict the dairy cow breeding industry, seriously endangers the health of cattle and hinders the development of the dairy cow and beef cow breeding industry. The factors causing calf diarrhea are complicated, wherein diarrhea caused by infectious agents is the most serious and is often mixed, bovine rotavirus, bovine coronavirus and escherichia coli K99 are important pathogens causing calf diarrhea, and the diarrhea caused by the three pathogens has similar clinical symptoms, acute onset and easy mixed infection; because the differential diagnosis method is not adopted or the diagnosis is not in time and other factors are not adopted, the diarrhea of the calves can not be treated by symptoms, the incidence rate of the diarrhea of the calves reaches 70 percent, the death rate reaches 50 to 90 percent, and huge economic losses are caused for the dairy cows and beef cattle breeding industry.
Bovine rotavirus infection is an acute gastrointestinal infectious disease of various calves caused by rotavirus (Bovine Rotavirus, BRV), and BRV can persist in infected cattle, showing high infection rate; BRV infection can cause calf mental depression, anorexia, emesis, diarrhea, dehydration and other clinical symptoms. BRV is more resistant to external factors and remains infectious for half a year in faeces and milk without antibodies. The feces of patients and recessive infected patients contain a large amount of rotavirus and can be transmitted to susceptible people and animals through the alimentary canal. The disease mainly occurs in calves, and the age of the onset of the disease is mainly 1-10 days. The disease is more serious in spring and autumn. In calves suffering from diarrhea, a combined infection of various pathogens is often observed, wherein 27-36% of bovine rotavirus is the main cause of calf diarrhea, and death is often caused by mixed feeling of bovine coronavirus and escherichia coli.
Bovine coronavirus (Bovine coronavirus, BCoV) is one of the most prominent pathogens responsible for adult Niu Dong dysentery and neonatal calf diarrhea (blood and mucus are common in diarrhea feces), and infections of BCoV are widely distributed around the world. BCoV belongs to the order of the nested viridae, the family of coronaviridae, and the genus betacoronaviridae. BCoV can cause diseases including calf diarrhea (CALF DIARRHEA, CD) and adult Niu Dongli (WINTER DYSENTERY of add cattle, WD) and respiratory diseases. The calf coronavirus disease is usually at 1-90 days old, diarrhea usually occurs at 1-2 weeks old, and is very similar to rotavirus infection, and the two are easy to mix. BCoV has multiple outbreaks, resulting in higher mortality for newborn calves. Enterotoxin Type Escherichia Coli (ETEC) is one of the common and important pathogenic bacteria for diarrhea of young animals, and such escherichia coli can adhere to small intestinal mucosa of animals by means of the generated pilus antigen, colonize and generate enterotoxin, thereby exhibiting pathogenic effects. The E.coli pilus antigens that have been discovered and studied in various animals today are mainly K99, K88, F41, etc., where K99 is the main pathogenic antigen.
The current BRV, BCoV and ETEC K99 diagnosis methods mainly comprise pathogen separation, conventional RT-PCR, multiplex PCR, ELISA, loop-mediated isothermal amplification and other methods, but the detection methods have obvious limitations. In order to overcome the defects of the conventional PCR in the aspects of sensitivity, efficiency, pathogen content measurement and the like, a method capable of detecting the three pathogens simultaneously is needed to be established, technical support is provided for prevention and control of calf diarrhea diseases, and the healthy development of the cattle industry is ensured to be protected.
Disclosure of Invention
The invention provides a triple fluorescence quantitative PCR detection method for main pathogens of calf diarrhea and application thereof, which solves the problems of insufficient sensitivity, efficiency, pathogen content measurement and the like of conventional PCR.
The technical scheme of the invention is as follows:
The method uses RNA or DNA of a sample to be detected as a template, selects an N gene of BCoV, an NSP5 gene of BRV and a K99 gene of escherichia coli as detection target genes, screens and determines a candidate region of a gene sequence to be amplified by comparing target gene sequences, designs quantitative PCR amplification primers according to the determined gene sequences, designs probes in the middle of the designed amplification primers, performs fluorescent quantitative PCR detection, and finally analyzes a fluorescent quantitative PCR amplification curve and Ct value to obtain detection results;
The main pathogens of calf diarrhea are bovine rotavirus, bovine coronavirus and escherichia coli K99;
The amplification primers of the fluorescent quantitative PCR comprise a BRV PCR amplification primer pair BRV-NSP5-U, BRV-NSP5-L2, a BCoV PCR amplification primer pair BCoV N476F, a BCoV N570R and an escherichia coli K99 amplification primer pair K99-U1 and K99-L2; the BRV-NSP5-U is shown as SEQ ID NO.1, the BRV-NSP5-L2 is shown as SEQ ID NO.2, the BCoV N476F is shown as SEQ ID NO.4, the BCoV N570R is shown as SEQ ID NO.5, the K99-U1 is shown as SEQ ID NO.7, and the K99-L2 is shown as SEQ ID NO. 8.
Further, the probes comprise probes BRV-NSP5-P2 corresponding to a BRV PCR amplification primer pair, probes BCoV N502P corresponding to a BCoV PCR amplification primer pair and probes K99-P2 corresponding to an E.coli K99 amplification primer pair; the BRV-NSP5-P2 is shown as SEQ ID NO.3, the BCoV N502P is shown as SEQ ID NO.6, and the K99-P2 is shown as SEQ ID NO. 9.
Further, the detection method adopts a fluorescence quantitative PCR system of 20 mu L, wherein the fluorescence quantitative PCR system comprises 5.4 mu L of water, 2X One StepU Mix mu L, one StepU Enzyme MIX mu L of water, 0.1 mu L of BRV-NSP5-U, BRV-NSP5-L2, 0.1 mu L of BRV-NSP5-P2, 0.1 mu L of BCoV N476F and 0.1 mu L of BCoV N570R, 0.1 mu L of BCoV N502P, 0.2 mu L of K99-U1, 0.2 mu L of K99P 2, 0.4 mu L of ROX II reference dye and 2 mu L of template.
Furthermore, the fluorescent quantitative PCR amplification condition adopted by the detection method is 15min at 50 ℃;95℃30s,95℃10s,55℃50s,45 cycles.
The primer in the kit is a BRV PCR amplification primer pair BRV-NSP5-U, BRV-NSP5-L2, a BCoV PCR amplification primer pair BCoV N476F, a BCoV N570R and an escherichia coli K99 amplification primer pair K99-U1 and K99-L2; the BRV-NSP5-U is shown as SEQ ID NO.1, the BRV-NSP5-L2 is shown as SEQ ID NO.2, the BCoV N476F is shown as SEQ ID NO.4, the BCoV N570R is shown as SEQ ID NO.5, the K99-U1 is shown as SEQ ID NO.7, and the K99-L2 is shown as SEQ ID NO. 8; the probes in the kit are probes BRV-NSP5-P2 corresponding to a BRV PCR amplification primer pair, probes BCoV N502P corresponding to a BCoV PCR amplification primer pair and probes K99-P2 corresponding to an E.coli K99 amplification primer pair; the BRV-NSP5-P2 is shown as SEQ ID NO.3, the BCoV N502P is shown as SEQ ID NO.6, and the K99-P2 is shown as SEQ ID NO. 9.
The kit is applied to detection of one or two or three pathogens of bovine rotavirus, bovine coronavirus and escherichia coli K99.
The beneficial effects of the invention are as follows:
(1) The detection method is a one-step fluorescence quantitative PCR method, is simple to operate and saves detection time.
(2) The invention can detect three pathogens of BRV, BCoV and escherichia coli K99 in one reaction system at the same time, obviously shortens the detection time, saves the cost while improving the detection efficiency, and has good application prospect.
(3) The detection primer set is designed aiming at BRV, BCoV and escherichia coli K99 conserved gene sequences, and is used for detecting bovine parvovirus, bovine viral diarrhea virus, bovine enterovirus, salmonella and clostridium, and no strip is amplified, so that the specificity of the primer set is strong.
(4) The invention has high detection sensitivity to BRV, BCoV and escherichia coli K99, and can reach 10 copies/mu L.
(5) The detection method of the invention is obviously superior to the conventional RT-PCR method, and improves the sensitivity of detection.
(6) The detection method of the invention has stable batch-to-batch repetition and batch-to-batch repetition, and is suitable for clinical large-scale detection.
Drawings
FIG. 1 shows amplification curves (A-C) and standard curves (D-F) for bovine rotavirus, bovine coronavirus and E.coli K99 plasmid standards;
FIG. 2 shows a triple PCR amplification curve (A) and a standard curve (B);
FIG. 3 shows the results of triple fluorescence quantitative PCR specific detection;
FIG. 4 is a comparison of RT-PCR with triple fluorescent quantitative PCR detection results, wherein A is the comparison of qPCR and RT-PCR detection results of 1100 diarrhea samples; b is the electrophoresis result after BRV RT-PCR; c is the electrophoresis result after BCoV RT-PCR; d is the result of electrophoresis after K99 RT-PCR.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will now be clearly and completely described, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, one of ordinary skill in the art would be able to make no inventive effort
All other embodiments obtained fall within the scope of protection of the present invention.
Test materials used in the examples:
The plasmid extraction Kit is Gene JET PLASMID MINI PREP KIT (Thermo Scientific), the gel recovery Kit hey Gene JET Gel Extraction Kit (Thermo Scientific), the RNA extraction Kit EasyPure Viral DNA/RNA Kit (ER 201) is purchased from Beijing full gold Biotechnology Co., ltd., hiScripto II U One Step qRT-PCR Probe Kit (Q223) is purchased from Norvirzan Biotechnology Co., ltd.
Example 1
1.1 Primer and probe design.
The sequences were aligned by MegAlign with reference to the genomic sequences recorded in GenBank, and the N gene of BCoV, the NSP5 gene of BRV, and the E.coli K99 gene were selected as detection target genes. Further comparing target gene sequences, determining candidate regions of the gene sequences to be amplified, designing quantitative PCR amplification primers according to the determined gene sequences, designing probe primers in the middle of the designed amplification primers, and synthesizing the quantitative PCR primers and probes by Jilin Kumei biotechnology company, wherein the sequences are as follows:
TABLE 1 primer and probe sequences
1.2 Preparation of plasmid Standard.
(1) Amplification of fragments of interest
Extracting viral and bacterial genomes respectively by EasyPure Viral DNA/RNA Kit, and using the viral or bacterial genomes as templates, amplifying the target fragments by using the above primers according to a 25 mu L PCR system (2 xTaq PCR Mix 10 mu L, 10mM upper and downstream primers 1 mu L each, viral cDNA/bacterial DNA 1 mu L, and ddH 2 O12 mu L) under the following amplification conditions: 95 ℃ 5 min; 30 s at 95 ℃,30 s at 53 ℃,30 s at 72 ℃ and 30 cycles; 10min at 72 ℃;30 min at 4 ℃. After amplification, the PCR product was subjected to 1% agarose gel electrophoresis detection and analysis, and the target fragment was purified using a gel recovery kit.
(2) Ligation transformation
The above gel recovery product was ligated to pMD18-T vector (TaKaRa Co.) and the ligation product was added to DH 5. Alpha. Competent cells (ice bath for 30 min;42℃heat shock 90 s; ice bath 2 min). The bacterial liquid was transferred to a non-resistant LB medium, and cultured under shaking at 37℃and 220 rpm for 45 min. mu.L of the bacterial liquid was uniformly spread on an LB plate containing ampicillin, and cultured overnight at a constant temperature of 37 ℃.
(3) Screening and identification of Positive clones
The monoclonal colony on the LB plate was picked up, added to 5mL of LB medium with ampicillin resistance, and cultured with shaking at 37℃and 220 rpm for 12 hours. And (3) performing bacterial liquid PCR verification (amplifying the corresponding target fragment) and sequencing by using the corresponding primers, and extracting plasmids for standby by using a plasmid DNA small extraction kit. The identified positive recombinant plasmids were designated pMD-NSP, pMD-N, pMD-K99. Plasmid concentrations were determined using a NanoDrop instrument, and the concentrations of pMD-NSP and pMD-N, pMD-K99 recombinant plasmids were 236 ng/. Mu.L, 254 ng/. Mu.L and 228 ng/. Mu.L, respectively, and the copy numbers of the recombinant plasmids were 7.7X10 10 copies/. Mu.L, 8.32X10 10 copies/. Mu.L and 7.26X10 10 copies/. Mu.L, respectively, as plasmid standards, by formula conversion.
The calculation formula of the copy number is as follows: copy number = plasmid concentration x 6.02 x 10 23/(660 x total plasmid length).
1.3 Single fluorescent quantitative PCR method.
Total RNA was extracted from the sample using EasyPure Viral DNA/RNA Kit, and fluorescent quantitative PCR was performed using HiScripto II U One Step qRT-PCR Probe Kit with a reaction system of 20. Mu.L, 6.1. Mu.L of water, 2X One StepU Mix. Mu.L, one StepU Enzyme MIX. Mu.L, 0.2. Mu.L of each of the upstream and downstream primers, 0.1. Mu.L of the Probe, 0.4. Mu.L of the ROX II reference dye, and 2. Mu.L of the template. The fluorescent quantitative PCR reaction program is as follows: 50 ℃ for 15min;95℃30s,95℃10s,55℃50s,45 cycles.
In order to draw a standard curve of single fluorescent quantitative PCR amplification, three plasmid standards are subjected to 10-fold serial dilution (10 0、101、102、103、104、105、106 and 10 7 copies/. Mu.L 7 dilutions) as templates for amplification, negative control is set at the same time, and the three plasmid standards are placed on a Applied Biosystems QuantStudio 5 real-time fluorescent quantitative PCR instrument for fluorescent quantitative PCR reaction to obtain an amplification curve, and the correlation coefficients R 2 of the amplification curves are analyzed by software to be respectively: 0.995 (BRV), 0.990 (BCoV), 0.997 (K99), has good linear relationship, meets the requirement of standard curve (R2 > 0.99), and the established standard curve is shown in figure 1 .BRV：Y=-3.266 log(X)+36.726 R2= 0.995 Eff% = 102.388,BCoV: Y=-3.252 log(X)+38.598 R2=0.990 Eff% = 102.994, K99: Y=-3.558 log(X)+ 38.143 R2=0.997 Eff% = 91.007
1.4 Triple fluorescence quantitative PCR method.
Fluorescent quantitative PCR was performed using HiScripto II U One Step qRT-PCR Probe Kit in a reaction system of 20. Mu.L, 5.4. Mu.L of water, 2X One StepU Mix. Mu.L, one StepU Enzyme MIX. Mu.L, 0.1. Mu.L each of BRV primer NSP5-U/NSP5-L2, 0.1. Mu.L each of P2 Probe, 0.1. Mu.L each of BCoV primer N476F/N570R, 0.1. Mu.L each of N502P Probe, 0.2. Mu.L each of K99 primer U1/L2, 0.2. Mu.L each of K99P 2 Probe, 0.4. Mu.L each of ROX II reference dye, and 2. Mu.L each of template. The triple fluorescence quantitative PCR reaction procedure was: 15 min at 50 ℃;95℃for 30 s,95℃for 10s, 55℃for 50 s,45 cycles.
In order to draw a standard curve of triple fluorescence quantitative PCR amplification, three prepared plasmid standard substances are diluted according to a 10-time gradient, fluorescent quantitative PCR amplification is respectively carried out after a concentration interval is 10 8-101 copies/. Mu.L, a fluorescent signal can be detected by the recombinant plasmid standard substances between the concentration of 10 8-101 copies/. Mu.L, the standard curve of the fluorescent quantitative PCR is drawn by taking the logarithm of the copy number as an ordinate and the Ct value as an abscissa through analysis of automatic analysis software of a system, and the result is shown in a figure 2, wherein the figure 2A is an amplification curve of three plasmids, and the figure 2B is a standard curve of three plasmids. As shown in the figure, the amplification efficiency of the standard products constructed by BRV, BCoV and K99 in the range of 10 8-101 copies/. Mu.L is more than 87%, and the correlation coefficient R 2 is more than 0.99, which indicates that the initial template number and Ct value of each standard product show good linear relation.
The linear equation of the resulting copy number (x) and Ct value (y) is:
BCoV: Y=-3.468 log(X)+42.822R2=0.994Eff%=94.227,
BRV: Y=-3.657 log(X)+41.093R2=0.998Eff%=87.680,
K99: Y=-3.289 log(X)+ 39.259R2=0.993Eff%=101.388。
Sensitivity test:
Triple fluorescence quantitative PCR was performed with serial 10-fold dilutions of BRV and BCoV virus solution, resulting in a minimum detectable BRV and BCoV of 0.05 TCID 50/mL and 0.14 TCID 50/mL. The K99 bacterial liquid is serially diluted by 10 times, and triple fluorescence quantitative PCR is carried out, wherein the lowest detectable K99 is 0.7433 cfu/mL.
Three plasmid standards were serially diluted 10-fold and subjected to triple fluorescence quantitative PCR, and as a result, 10 copies were detected. The results show that the BRV, BCoV and K99 triple fluorescence quantitative PCR has high sensitivity, and can be used for clinical sample detection.
Specificity test:
The method is characterized in that BRV, BCoV and K99 standard substances are used as positive control, bovine Parvovirus (BPV), bovine Enterovirus (BEV) Bovine Viral Diarrhea Virus (BVDV), salmonella (Salmonella), clostridium (Clostridium) and water are used as samples, total DNA/RNA is extracted as a template of fluorescent quantitative PCR, fluorescent quantitative PCR amplification is carried out according to the established optimal reaction conditions, and as a result, as shown in figure 3, the respective amplification curves can be detected by the three standard substances respectively, and no amplification curve appears in other pathogens and negative control, so that the method has good specificity.
Repeatability test:
The BRV, BCV, K99 recombinant plasmids were mixed in equal proportions after 10-fold serial dilutions, and 4 concentration gradients (final concentrations of 10 6 copies/. Mu.L, 10 5 copies/. Mu.L, 10 4 copies/. Mu.L, and 10 3 copies/. Mu.L) were used as plasmid mixtures, while setting water as a negative control. Repeating the detection for 3 times by using the established fluorescence quantitative PCR method, and performing an in-group repeatability test; the same concentration standard diluted for 3 times in different time periods was subjected to repeated experiments between groups, the experiment results were counted, and the repeatability of the method was evaluated, and the results are shown in table 2. As shown in Table 2, the intra-group variation coefficient is less than 4% and the inter-group variation coefficient is less than 2%, which indicates that the multiplex fluorescence quantitative detection method established by the invention has good repeatability.
TABLE 2 reproducible results of multiplex fluorescent quantitative PCR
Comparison with conventional PCR:
as can be seen from FIG. 4, the sensitivity of multiplex fluorescent quantitative PCR is 10-100 times higher than that of conventional RT-PCR.
Example 2
And (5) detecting clinical samples.
And detecting the collected 1100 clinical calf diarrhea stool samples by using the established triple fluorescence quantitative PCR method. Total RNA was extracted from the sample using EasyPure Viral DNA/RNA Kit, and fluorescent quantitative PCR was performed using HiScripto II U One Step qRT-PCR Probe Kit with a reaction system of 20. Mu.L: 5.4. Mu.L of water, 2X One StepU Mix. Mu.L, one StepU Enzyme MIX. Mu.L, 0.1. Mu.L each of BRV primer NSP5-U/NSP5-L2, 0.1. Mu.L each of P2 probe, 0.1. Mu.L each of BCoV primer N476F/N570R, 0.2. Mu.L each of N502P probe, 0.2. Mu.L each of K99 primer U1/L2, 0.2. Mu.L each of K99P 2 probe, 0.4. Mu.L each of ROX II reference dye, and 2. Mu.L each of template. The triple fluorescence quantitative PCR reaction procedure was: 50 ℃ for 15min;95℃30s,95℃10s,55℃50s,45 cycles. Analysis of fluorescent quantitative PCR amplification curves and Ct values shows that most northern areas in China have certain popularity, wherein the BRV positive rate of Xinjiang Uygur autonomous region and Jiangsu province is higher, and the BRV positive rate is not detected in Hebei and Qinghai due to fewer samples. The Heilongjiang province is used as the north province of China, and the BRV and BCoV positive rates are high. From the results, the co-infection cases are more, wherein 86 clinical samples are infected by BRV+BCoV; 29 parts of BRV+K99 mixed infection; 28 parts of BCoV+K99 mixed infection; 8 parts of mixed infection of three diseases.
TABLE 3 detection results of clinical samples in different regions
The fluorescent quantitative PCR method has 10-100 times higher sensitivity than RT-PCR, and the RT-PCR can not detect the condition of low pathogen content, and is easy to cause false negative, thus leading to misdiagnosis and affecting the prevention and control of the disease. In addition, the fluorescent quantitative PCR detection method is short in time, the result can be judged in 1h and 23min, the detection time of the RT-PCR detection method is about 2h, the electrophoresis time is 20-30 min, and the fluorescent quantitative PCR is saved by about 1h compared with the RT-PCR. More importantly, the triple fluorescence quantitative PCR method can detect pathogens of three calf diarrhea simultaneously, is suitable for high-throughput rapid diagnosis, and provides a guarantee for preventing and controlling calf diarrhea and reducing economic loss.
The specific embodiments described above are only for the purpose of illustrating the invention and are not to be construed as limiting the invention. Obvious variations or modifications which extend from the spirit of the present invention are within the scope of the present invention.
Claims (5)
1. The method is characterized in that RNA or DNA of a sample to be detected is used as a template, an N gene of BCoV, an NSP5 gene of BRV and a K99 gene of escherichia coli are selected as detection target genes, target gene sequences are compared, candidate regions of the gene sequences to be amplified are determined, quantitative PCR amplification primers are designed according to the determined gene sequences, probes are designed in the middle of the designed amplification primers, fluorescent quantitative PCR detection is carried out, and finally fluorescent quantitative PCR amplification curves and Ct values are analyzed to obtain detection results;
The main pathogens of calf diarrhea are bovine rotavirus, bovine coronavirus and escherichia coli K99;
The amplification primers of the fluorescent quantitative PCR comprise a BRV PCR amplification primer pair BRV-NSP5-U, BRV-NSP5-L2, a BCoV PCR amplification primer pair BCoV N476F, a BCoV N570R and an escherichia coli K99 amplification primer pair K99-U1 and K99-L2; the BRV-NSP5-U is shown as SEQ ID NO.1, the BRV-NSP5-L2 is shown as SEQ ID NO.2, the BCoV N476F is shown as SEQ ID NO.4, the BCoV N570R is shown as SEQ ID NO.5, the K99-U1 is shown as SEQ ID NO.7, and the K99-L2 is shown as SEQ ID NO. 8;
The probes comprise probes BRV-NSP5-P2 corresponding to a BRV PCR amplification primer pair, probes BCoV N502P corresponding to a BCoV PCR amplification primer pair and probes K99-P2 corresponding to an E.coli K99 amplification primer pair; the BRV-NSP5-P2 is shown as SEQ ID NO.3, the BCoV N502P is shown as SEQ ID NO.6, and the K99-P2 is shown as SEQ ID NO. 9.
2. The method of claim 1, wherein the method employs a fluorescent quantitative PCR system of 20. Mu.L, which comprises water of 5.4. Mu.L, 2X One StepU Mix. Mu.L, one StepU Enzyme MIX. Mu.L, BRV-NSP5-U, BRV-NSP5-L2 of 0.1. Mu.L each, BRV-NSP5-P2 of 0.1. Mu.L, BCoV N476F, BCoV N570R of 0.1. Mu.L each, BCoV N502P of 0.1. Mu.L, K99-U1, K99-L2 of 0.2. Mu.L each, K99P 2.2. Mu.L, ROX II reference dye of 0.4. Mu.L, and template of 2. Mu.L.
3. The method according to claim 1, wherein the fluorescent quantitative PCR amplification conditions used in the method are 15min at 50 ℃;95℃30s,95℃10s,55℃50s,45 cycles.
4. The kit is characterized in that primers in the kit are BRV PCR amplification primer pairs BRV-NSP5-U, BRV-NSP5-L2, BCoV PCR amplification primer pairs BCoV N476F, BCoV N570R and escherichia coli K99 amplification primer pairs K99-U1 and K99-L2; the BRV-NSP5-U is shown as SEQ ID NO.1, the BRV-NSP5-L2 is shown as SEQ ID NO.2, the BCoV N476F is shown as SEQ ID NO.4, the BCoV N570R is shown as SEQ ID NO.5, the K99-U1 is shown as SEQ ID NO.7, and the K99-L2 is shown as SEQ ID NO. 8; the probes in the kit are probes BRV-NSP5-P2 corresponding to a BRV PCR amplification primer pair, probes BCoV N502P corresponding to a BCoV PCR amplification primer pair and probes K99-P2 corresponding to an E.coli K99 amplification primer pair; the BRV-NSP5-P2 is shown as SEQ ID NO.3, the BCoV N502P is shown as SEQ ID NO.6, and the K99-P2 is shown as SEQ ID NO. 9.
5. The use of the kit of claim 4 for the preparation of a product for detecting one or both of bovine rotavirus, bovine coronavirus and escherichia coli K99.
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