CN114774588A

CN114774588A - Primer and probe combination for simultaneously detecting three bovine infectious pathogens

Info

Publication number: CN114774588A
Application number: CN202210550478.6A
Authority: CN
Inventors: 郭爱珍; 马耀争; 胡长敏; 陈颖钰; 朱杰; 陈曦; 陈建国
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2022-07-22

Abstract

The invention discloses a primer and probe combination for triple real-time fluorescent quantitative PCR (Multiplex real-time fluorescent polymerase chain reaction) for simultaneously detecting Bovine Viral Diarrhea Viruses (BVDV), Bovine Rotavirus (BRV) and Bovine Coronavirus (BCV), wherein the primer and probe are specifically directed at a 5' UTR gene fragment of the BVDV virus, a VP6 gene fragment of the BRV virus and an N gene fragment of the BCV virus, and the nucleotide sequence is shown as SEQ ID Nos. 1-9. The invention can realize qualitative or quantitative detection of three viruses only by carrying out PCR amplification on a sample once, and has the advantages of simple and convenient operation, strong specificity, high sensitivity, good repeatability and the like.

Description

Primer and probe combination for simultaneously detecting three bovine infectious pathogens

Technical Field

The invention belongs to the field of molecular detection, and particularly relates to a primer and probe combination for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus, and application of the primer and probe combination.

Technical Field

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of Bovine viral diarrhea, and BVDV virus infection shows a wide range of clinical manifestations including acute bleeding syndrome, long-lasting viral infection, decreased immunity, and severe respiratory and gastrointestinal lesions; of these, reproductive disorders following BVDV infection are among the most serious consequences. After the BVDV virus is infected, persistent infection is mostly presented, a part of infected cattle are healthy in appearance, antibodies are negative, but the antigens of the virus are positive, and the persistently infected cattle carry the virus for the whole life, so that the health of the cattle herd is greatly influenced, and the cattle industry is greatly lost.

Bovine Rotavirus (BRV) belongs to the genus Rotavirus (Rotavirus) of the reoviridae family (reoviridae). The rotavirus mainly infects calves within 1 month of age, wherein the calves within 1 week of age are most susceptible, after BRV infection, sick animals are listened, salivated, unwilling to stand and accompanied with severe watery diarrhea, the tail part of the calves is infected with yellow watery excrement, secondary germ transmission is caused by the reduction of the body resistance, and the mixed infection causes the increase of acute diarrhea cases of the calves and higher death rate. Moreover, different serotypes of BRV generate gene recombination or gene transfer in a certain area, so that the infection of bovine rotavirus is more serious, and the economic benefit of animal husbandry is greatly influenced.

Bovine Coronavirus (BCV) belongs to the family coronaviridae, belongs to subgroup 2a, and is mainly responsible for diarrhea of newborn calves, and is also an important pathogen of winter dysentery and respiratory tract infection of adult cattle. Wherein the calf diarrhea is mostly developed at 8-10 d age, the incubation period is generally 1-2 d, and the calf diarrhea can die due to death and has higher death rate. After the calf is infected with BCV, the calf is depressed in spirit, slight high fever occurs at the initial stage, watery diarrhea occurs suddenly later, the excrement is light yellow to milky white, then the calf is quickly dehydrated and accompanied with inflammatory digestive tract lesions, and even a large amount of blood or blood clots occur in the excrement; adult cattle develop winter diarrhea after infection, and new dairy cows have symptoms of sudden acute diarrhea and a sharp decline in milk yield. Moreover, BCV can cause persistent infection of cattle, is asymptomatic and carries toxicity for a long time, and poses great threat to the health of cattle herds.

Meanwhile, with the development of cattle raising industry, the large-scale and intensive degree is higher and higher, the calf diarrhea cases caused by BVDV, BRV and BCV mixed infection are remarkably increased, and great loss is caused to the cattle raising industry. Therefore, the establishment of a laboratory detection method which is rapid, sensitive, high in sensitivity and good in specificity aiming at BVDV, BRV and BCV has very critical significance for early clinical diagnosis and implementation of clinical treatment, and can also be used for pathogen epidemiological research of calf diarrhea. At present, the main diagnostic methods of the three viruses comprise virus separation and identification, serum neutralization test, ELISA technology, PCR, real-time fluorescence quantitative PCR technology and the like. The separation and identification of the virus is a gold standard, but the method is complex to operate and takes longer time for diagnosis; the serum neutralization test can be used for detecting virus antigens, has stronger specificity, but has complex operation and longer time requirement; the ELISA technology can also be used for detecting virus antigens, is simple and rapid to operate, has stronger specificity, lacks a commercial kit and has lower diagnostic sensitivity; the traditional PCR technology cannot quantify the DNA of the template, and finally, the DNA can be interpreted through gel electrophoresis, so that the operation is relatively long, and the pollution risk is high; the real-time fluorescent quantitative PCR technology combines the fluorescent substance with the traditional PCR technology, not only can accurately determine the nature and the absolute quantity of pathogens, but also has simple operation, good specificity and high sensitivity, and can dynamically research the reactivation or persistent infection of potential pathogens in the whole course of disease; the multiplex real-time fluorescent quantitative PCR technology can accurately and quickly detect mixed infection caused by various pathogens. Therefore, the purpose of the research is to design primers and probes on the basis of a real-time fluorescent quantitative PCR technology, establish a group of multiplex real-time fluorescent quantitative PCR methods aiming at BVDV, BRV and BCV, and be used for laboratory virus rapid screening and identification of BVDV, BRV and BCV, so as to provide a rapid etiology diagnosis method for calf diarrhea.

Disclosure of Invention

The invention aims to provide a multiplex real-time fluorescent quantitative PCR primer and probe combination for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus and a detection method.

The above purpose is realized by the following technical scheme:

a multiplex real-time fluorescent quantitative PCR primer and probe combination for the simultaneous detection of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus wherein:

the nucleotide sequence of an upstream primer designed aiming at the bovine viral diarrhea virus is shown as SED ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3; the nucleotide sequence of the upstream primer designed aiming at the bovine rotavirus is shown as SED ID NO. 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5, and the probe sequence is shown as SEQ ID NO. 6; the nucleotide sequence of the upstream primer designed aiming at the bovine coronavirus is shown as SED ID NO. 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8, and the probe sequence is shown as SEQ ID NO. 9: the method comprises the following specific steps:

the 5 'end of the probe is connected with a fluorescent group, and the 3' end of the probe is connected with a fluorescence quenching group. Wherein the fluorescent group of SEQ ID NO. 3 is HEX, and the fluorescence quenching group is BHQ 1; the fluorescent group of SEQ ID NO. 6 is ROX, and the fluorescence quenching group is BHQ 2; the fluorescent group of SEQ ID NO. 9 is CY5, and the fluorescence quenching group is BHQ 3.

The primers can amplify a 151bp long sequence of the 5' UTR gene region (GenBank accession number: LC099927.1) of BVDV, and the sequence is shown as SEQ ID No. 10. A 169bp long sequence of the VP6 gene region of BRV (GenBank accession AB374146.1), which is shown in SEQ ID No. 11. A 191bp long sequence of the N gene region of BCV (GenBank accession number: LC494138.1), which is shown in SEQ ID No. 12.

The primer and probe combination can be applied to laboratory screening and identification of bovine viral diarrhea viruses, bovine rotavirus and bovine coronavirus, and can also be applied to clinical diagnosis of related diseases such as calf diarrhea. Because the combination of the multiplex real-time fluorescent quantitative PCR primer and the probe is provided, when identification or diagnosis is carried out, qualitative or quantitative detection of the three viruses can be realized only by carrying out PCR amplification on a sample once.

The primer and probe combination can be used for preparing a multiplex real-time fluorescent quantitative PCR detection kit for detecting bovine viral diarrhea viruses, bovine rotavirus and bovine coronavirus.

A multiplex real-time fluorescent quantitative PCR detection kit for detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus comprises the primer and probe combination.

The invention further provides a method for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus for non-diagnostic purposes, which comprises the step of adding the primer and the probe set into a reaction system and carrying out multiple real-time fluorescent quantitative PCR amplification.

The reaction system is as follows: 2 XT 5 Fast qPCR Mix 12.5. mu.L; 0.7 mu L of upstream primer and 1.0 mu L of downstream primer and probe for detecting the bovine viral diarrhea virus respectively; 0.8 mu L of upstream primer and 0.9 mu L of downstream primer and probe for detecting bovine rotavirus respectively; 0.8 muL of upstream primer and 1.0 muL of downstream primer and probe for detecting the bovine coronavirus respectively; 2 mu L of template DNA; make up to 25 μ L with double distilled water.

The reaction procedure of the amplification is as follows: 5min at 95 ℃; annealing at 95 ℃ for 10s and 60 ℃ for 30s for 42 cycles, and fluorescence signals were automatically collected at the end of each cycle.

The invention has the beneficial effects that:

the present invention is based on the BVDV-5' UTR; BRV-VP 6; specific primers and probes are designed and screened in the BCV-N gene region, a method for performing triple real-time fluorescent quantitative PCR detection on BVDV, BRV and BCV is established, and no report for performing PCR detection on the three viruses at the same time exists at present. Compared with the common qPCR, the method established by the invention has greatly improved detection efficiency. The invention further improves the accuracy and sensitivity of detection by screening a large number of detection conditions, realizes the qualitative and accurate quantification of BVDV, BRV and BCV, and has good development prospect and wide application space.

Drawings

FIG. 1: the experimental result of the sensitivity of the BVDV single-detection real-time fluorescent quantitative PCR is 1 multiplied by 10 from left to right in sequence⁷～1×10⁰Copies/. mu.L.

FIG. 2 is a schematic diagram: the BRV single-detection real-time fluorescence quantitative PCR sensitivity experiment result is 1 multiplied by 10 from left to right⁷～1×10⁰Copies/. mu.L.

FIG. 3: the BCV single-detection real-time fluorescence quantitative PCR sensitivity experiment result is 1 multiplied by 10 from left to right in sequence⁷～1×10⁰Copy/. mu.L.

FIG. 4: BVDV single-detection real-time fluorescence quantitative PCR standard curve.

FIG. 5: BRV single-detection real-time fluorescence quantitative PCR standard curve.

FIG. 6: BCV single-detection real-time fluorescence quantitative PCR standard curve.

FIG. 7 is a schematic view of: the experimental result of BVDV sensitivity in triple real-time fluorescent quantitative PCR is 1 multiplied by 10 from left to right⁷～1×10¹Copies/. mu.L.

FIG. 8: the experimental results of BRV sensitivity in triple real-time fluorescent quantitative PCR are 1 multiplied by 10 from left to right⁷～1×10¹Copies/. mu.L.

FIG. 9: the experimental result of BVDV sensitivity in triple real-time fluorescent quantitative PCR is 1 multiplied by 10 from left to right⁷～1×10¹Copies/. mu.L.

FIG. 10: triple real-time fluorescent quantitative PCR specificity experiment results. 1 is a BRV positive nucleic acid; 2 is a BVDV-positive nucleic acid; 3 is a BCV positive nucleic acid; 4-12 are negative controls and other viral nucleic acids, respectively.

FIG. 11: triple real-time fluorescent quantitative PCR standard curve.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Example 1 design of primers

Downloading BVDV-5' UTR from GenBank; BRV-VP 6; all nucleotide sequences of BCV-N gene region were compared and analyzed by MEGA software, and the gene sequence with the highest degree of conservation (BVDV GenBank accession No.: LC 099927.1; BRV GenBank accession No.: AB 374146.1; BCV GenBank accession No.: LC494138.1) and its specific region were selected, and specific primers and probes (Table 1) were designed and screened.

TABLE 1 primers and probes

Example 2 establishment and optimization of multiplex real-time fluorescent quantitative PCR method

1, establishment and optimization of single-detection real-time fluorescence quantitative PCR method for BVDV, BRV and BCV pathogens

1.1 Standard Positive plasmid preparation

BVDV-5' UTR was performed with the primers of Table 1; BRV-VP 6; a sequence of a BCV-N gene region (BVDV GenBank accession number: LC 099927.1; BRV GenBank accession number: AB 374146.1; BCV GenBank accession number: LC494138.1) including a primer sequence is inserted into a pUC57 vector to form recombinant plasmids which are respectively named as pUC57-5' UTR, pUC57-VP6 and pUC57-N as a standard positive plasmid template and a positive control in system construction.

1.2 establishment of Single-check real-time fluorescent quantitative PCR method

Single factor variables were set, and the primer concentration, probe concentration and annealing temperature were optimized for each single sample system using real-time fluorescent quantitative PCR with the primers and probes of Table 1. All optimization processes use standard positive plasmids as positive templates and double distilled water as negative templates. The template is amplified along with the real-time fluorescent quantitative PCR reaction, the fluorescence intensity is enhanced, the result can be analyzed through a fluorescence curve and a Ct value, a tube cover does not need to be opened in the whole process, the risk of aerosol pollution is reduced, and the optimal primer concentration, probe concentration and annealing temperature are selected according to the fluorescence signal intensity and the Ct value after the reaction is finished.

1.2.1 primer concentration optimization

Configuring a 25.00 mu L reaction system, setting 8 gradients (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 and 1.3 mu L) for the dosage (10 mu mol/L) of upstream and downstream primers of each pathogen, and fixing the dosage of the probe and the annealing temperature. And (4) judging according to the Ct value and the fluorescence signal intensity.

1.2.2 Probe concentration optimization

Configuring a 25.00 mu L reaction system, setting 8 gradients (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 and 1.3 mu L) for the dosage (10 mu mol/L) of each pathogen probe, taking the optimized result of 1.2.1 as the standard for the dosage of the primers, and fixing the annealing temperature. And (4) judging according to the Ct value and the fluorescence signal intensity.

1.2.3 temperature optimization

Configuring a 25.00 mu L reaction system, setting the annealing temperature at 56.5, 57, 57.5, 58, 58.5, 59, 59.5 and 8 annealing temperature gradients at 60 ℃ based on the optimized results of 1.2.1 and 1.2.2 for the primer dosage and the probe dosage (10 mu mol/L) of each pathogen. And (4) judging according to the Ct value and the fluorescence signal intensity.

The optimized single-detection real-time fluorescent quantitative PCR reaction system and the annealing temperature are shown in the table 2.

TABLE 2 optimization results of single-check real-time fluorescent quantitative PCR reaction system

Pathogens	Target genes	Amount of primer used (ul)	Amount of probe (μ l)	TM(℃)
					BVDV	5‘UTR	0.7	1.0	59
BRV	vp6	1.0	1.1	60
					BCV	N	0.8	1.2	58

Finally, the optimal system for determining the single-detection real-time fluorescent quantitative PCR reaction of each pathogen is shown in Table 3.

TABLE 3 pathogen single-detection real-time fluorescent quantitative PCR final reaction system

Reagent	Dosage of
		2×T5 Fast qPCR Mix(Probe)	12.5μL
10 mu mol of primer	BVDV 0.7. mu.L each; 1.0 μ L each of BRV; BCV 0.8. mu.L each
		10 μmol probe	BVDV 1.0μL；BRV 1.1μL；BCV 1.2μL
Template DNA	2μL
		Double distilled water	Make up to 25. mu.L

1.2.4 sensitivity analysis of real-time fluorescent quantitative PCR system for single detection of various pathogens

To determine the limit of detection (LOD) of the single-detection real-time fluorescent quantitative PCR system for each pathogen, we performed single-detection real-time fluorescent quantitative PCR reactions for each pathogen separately, using 10-fold serial dilutions of standard plasmids, and selected 1X 10⁰Copy/. mu.L, 1X 10¹Copy/. mu.L, 1X 10²And (3) taking the copied/mu L standard positive plasmid as a template, repeating each concentration for 20 times, considering the lowest concentration which meets the positive detection rate of more than or equal to 90 percent as reliable LOD, and carrying out a sensitivity experiment according to the optimization result of each pathogen single-detection real-time fluorescence quantitative PCR system.

Configuring 25.00 mu L of reaction systems according to the table 3, and respectively carrying out real-time fluorescence quantitative PCR experiments on the systems according to the temperature optimization result, wherein the program of each pathogen single-detection real-time fluorescence quantitative PCR amplification reaction is as follows: 5min at 95 ℃; 95 ℃ for 10s, TM (BVDV 59 ℃, BRV 60 ℃, BCV 58 ℃) for 30s, for a total of 42 cycles. The fluorescence signals are automatically collected at the end of each cycle, and the results show that the detection limit of single-sample systems of BVDV, BRV and BCV reaches 1 × 10⁰Copies/. mu.L, as in FIG. 1-3 and table 4.

TABLE 4 analysis sensitivity of pathogen single-detection real-time fluorescent quantitative PCR reaction system

1.2.5 construction of Standard Curve for real-time fluorescent quantitative PCR System for Individual detection of various pathogens

In order to verify the effectiveness and reliability of each pathogen single-detection real-time fluorescence quantitative PCR system. We performed real-time fluorescent quantitative PCR reaction on each single sample system, and selected the range of application from 1X 10⁷Copy/. mu.L to 1X 10⁰Copies/. mu.L of a 10-fold serial dilution of standard positive plasmid were used as template, 3 times for each concentration and a negative control was set. And drawing a standard curve according to the optimization result of each single detection system.

Configuring a 25.00 mu L reaction system according to the table 3, and respectively carrying out real-time fluorescence quantitative PCR experiments on each single-sample system according to the temperature optimization result, wherein the procedures of each single-sample real-time fluorescence quantitative PCR amplification reaction are as follows: 5min at 95 ℃; 95 ℃ for 10s, TM (BVDV 59 ℃, BRV 60 ℃, BCV 58 ℃) for 30s, for 42 cycles; the fluorescent signal was automatically collected at the end of each cycle. The results (FIGS. 4-6) show that: correlation coefficient R of BVDV, BRV and BCV²Respectively 0.996, 0.997 and 0.997, the amplification efficiencies E are respectively 103.2, 107.3 and 96.1 which are all between 90 and 110 percent, and a good linear relation is achieved; the regression equations of BVDV, BRV and BCV are respectively y₁＝-3.247x₁+43.278、y₂＝-3.159x₂+43.059 and y₃＝-3.369x₃+42.806, the results show that there is a good linear relationship between the initial template concentration and the threshold (Ct) value.

Establishment of 2BRV and BCV double real-time fluorescence quantitative PCR method

Preparing 25.00 μ L reaction system, mixing standard positive plasmids of BRV and BCV at equal ratio as template, setting 5 gradients for upstream and downstream primer dosage (10 μmol/L) of each pathogen and setting five gradients (such as BRV setting (0.8, 0.9, 1.0, 1.1, 1.2 μ L)) for the intermediate value of each single detection system; setting 5 gradients for the probe dosage of each pathogen by taking the optimized result of each single detection system as a median (10 mu mol/L); and 8 annealing temperature gradients were set: and the conditions of the double real-time fluorescence quantitative PCR are optimized at 56.5, 57, 57.5, 58, 58.5, 59, 59.5 and 60 ℃. The amplification program is 95 ℃ for 5 min; 10s at 95 ℃ and 30s TM for 42 cycles. The fluorescent signal was automatically collected at the end of each cycle.

The results after optimization are shown in table 5.

TABLE 5 optimization results of dual real-time fluorescent quantitative PCR reaction system

3. Establishment of triple real-time fluorescent quantitative PCR method

3.1 optimization of triple real-time fluorescent quantitation PCR reaction conditions

Preparing a 25.00 mu L reaction system, mixing standard positive plasmids of BVDV, BRV and BCV in equal proportion as a template, and taking the upstream and downstream primer dosage (10 mu mol/L) and probe dosage (10 mu mol/L) of BRV and BCV as references of the optimization result of the dual system; the primer amount and probe amount of BVDV were set (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 μ L) for 8 gradients, and 8 annealing temperature gradients were set: 56.5, 57, 57.5, 58, 58.5, 59, 59.5 and 60 ℃. And optimizing the reaction conditions of the triple real-time fluorescent quantitative PCR. The amplification program is 95 ℃ for 5 min; 10s at 95 ℃ and 30s TM for 42 cycles. The fluorescent signal was automatically collected at the end of each cycle.

The results after optimization are shown in table 6.

TABLE 6 optimization results of triple real-time fluorescent quantitative PCR reaction system

In summary, the optimal system for the triple real-time fluorescent quantitative PCR reaction was determined as shown in table 7.

TABLE 7 triple real-time fluorescent quantitative PCR final reaction system

Reagent	Amount of the composition
		2×T5 Fast qPCR Mix(Probe)	12.5μL
10 mu mol of primer	0.7. mu.L each of BVDV + 0.8. mu.L each of BRV + BCV: 0.8. mu.L each
		10. mu. mol probe	BVDV 1.0μL+BRV 0.9μL+BCV 1.0μL
Template DNA	2μL
		Double distilled water	Make up to 25 μ L

3.2 sensitivity analysis of triple real-time fluorescent quantitative PCR reaction System

In order to determine the limit of detection (LOD) of the triple real-time fluorescent quantitative PCR system on each pathogen, the triple real-time fluorescent quantitative PCR system is used for carrying out sensitivity experiments on each pathogen respectively, standard positive plasmids diluted by 10 times are used, and 1 × 10 times of standard positive plasmids are selected¹Copy/. mu.L, 1X 10²Copy/. mu.L, 1X 10³The standard positive plasmid of copy/. mu.L is used as a template, each concentration is repeated for 20 times, the lowest concentration which meets the positive detection rate of more than or equal to 90 percent is regarded as reliable LOD, and a sensitivity experiment is carried out according to the optimization result of a triple system.

Configuring a 25.00 mu L reaction system according to the table 7, and performing a real-time fluorescent quantitative PCR experiment on a triple system according to a temperature optimization result, wherein the procedure of the triple real-time fluorescent quantitative PCR amplification reaction is as follows: 5min at 95 ℃; 95 ℃ for 10s, TM60 ℃ for 30s, for a total of 42 cycles. The fluorescence signals are automatically collected at the end of each cycle, and the results show that the detection limit of the triple system on BVDV, BRV and BCV reaches 1 × 10¹Copies/. mu.L, as shown in FIGS. 7-9 and Table 8.

TABLE 8 analytical sensitivity of triple real-time quantitative PCR reaction System

3.3 specificity analysis of triple real-time fluorescent quantitative PCR reaction System

In order to determine the specificity of the triple real-time fluorescent quantitative PCR method and exclude false positive caused by other pathogens, DNA/cDNA of common calf diarrheal pathogens and other common pathogens including Escherichia coli F17, F41, K99, Salmonella, Cryptosporidium, IBR1, BPIV, BRSV and HM are taken as templates, BRV, BVDV and BCV are taken as positive controls, and the optimized triple system is used for detection and evaluation of the specificity. As shown in FIG. 10, only the positive control showed a typical amplification curve, and none of the other pathogens were amplified, indicating that the assay of the present invention has good specificity and can be used for specific detection of BVDV, BRV and BCV.

3.4 construction of Standard Curve of triple real-time fluorescent quantitative PCR reaction System

In order to verify the effectiveness and reliability of the triple real-time fluorescent quantitative PCR reaction system. We choose to use the range from 1 × 10⁷Copy/. mu.L to 1X 10⁰Copies/. mu.L of 10-fold serial dilutions of standard positive plasmid and their equal ratio mix as template, 3 times each concentration test and set negative control. And drawing a standard curve according to the optimization result of the triple system.

Configuring a 25.00 mu L reaction system according to the table 7, and carrying out real-time fluorescence quantitative PCR experiment on a triple system according to the temperature optimization resultThe procedure for the light quantitative PCR amplification reaction was: 5min at 95 ℃; 95 ℃ for 10s, TM60 ℃ for 30s, for a total of 42 cycles. Automatically collecting fluorescent signals at the end of each cycle; the results (fig. 11) show: correlation coefficient R of BVDV, BRV and BCV²Respectively 0.999, 0.999 and 0.998, the amplification efficiencies E are respectively 99.9, 102.0 and 92.2 which are all between 90 percent and 110 percent, and a good linear relation is achieved; the regression equations of BVDV, BRV and BCV are respectively y₁＝-3.324x₁+43.057、y₂＝-3.275x₂+42.960 and y₃＝-3.523x₃+43.862, the results show that there is a good linear relationship between the initial template concentration and the threshold (Ct) value.

3.5 repeatability experiment of triple real-time fluorescent quantitative PCR reaction system

To determine the reproducibility of this triple real-time fluorescent quantitative PCR method, we chose 1X 10³Copy/. mu.L, 1X 10⁴Copy/. mu.L, 1X 10⁵BVDV, BRV and BCV were subjected to an in-group and in-group repeat experiments within one week, each reaction repeated 3 times, by copying/μ L of a 10-fold serial dilution of standard positive plasmids of each pathogen and mixing them in equal proportions as templates. The Coefficient of Variation (CV) of the Cq values of the samples at each concentration in the experiment was calculated to evaluate their reproducibility. The results are shown in Table 9: the Coefficient of Variation (CVs) between the intragroup and intergroup for each pathogen in the triple combination was less than 3%. The triple real-time fluorescent quantitative PCR reaction system established by the research has good repeatability and high stability.

TABLE 9 results of the repeatability tests

Example 3 evaluation of clinical sample detection by triple real-time fluorescent quantitative PCR System

Clinical sample detection and coincidence rate analysis were performed on 143 cDNA samples stored in the laboratory using the triple real-time fluorescent quantitative PCR system established in this study and the general PCR detection method obtained by consulting literature (table 10). The sequencing verification of the result of each common PCR detection is carried out by Wuhan Kyok Biotechnology GmbH; the determination standard of the triple real-time fluorescent quantitative PCR detection method is as follows:

1. the Ct value of the detected sample is less than 40, and the sample is judged to be positive when a typical amplification curve appears;

2. the Ct value of the detected sample is more than 40, and the sample needs to be subjected to recheck when a typical amplification curve appears. If the rechecking result is positive, judging the rechecking result to be negative;

3. if there is no Ct value and no amplification curve, the result is judged to be negative.

The results are shown in Table 11: the detection rates of BVDV are 15/143 (10.48%) and 8/143 (5.59%) respectively, and the coincidence rate is 70%; the BRV detection rates are 17/143 (11.88%) and 11/143 (7.69%), and the coincidence rate is 78%; the BCV detection rates are 18/143 (12.58%) and 12/143 (8.39%) respectively, and the coincidence rate is 80%.

TABLE 10 primer sequences for control PCR

TABLE 11 clinical specimen test results

Sequence listing

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Claims

1. A multiplex real-time fluorescent quantitative PCR primer and probe combination for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus, characterized in that the primer and probe combination is as follows:

the upstream primer sequence for detecting the bovine viral diarrhea virus is shown as SED ID NO.1, the downstream primer sequence is shown as SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;

the upstream primer sequence for detecting the bovine rotavirus is shown as SED ID NO. 4, the downstream primer sequence is shown as SEQ ID NO. 5, and the probe sequence is shown as SEQ ID NO. 6;

the upstream primer sequence for detecting bovine coronavirus is shown as SED ID NO. 7, the downstream primer sequence is shown as SEQ ID NO. 8, and the probe sequence is shown as SEQ ID NO. 9.

2. The multiplex real-time quantitative fluorescent PCR primer and probe combination according to claim 1, wherein the probe is labeled with a fluorescent group at the 5 'end and a fluorescence quenching group at the 3' end.

3. The multiplex real-time fluorescent quantitative PCR primer and probe combination according to claim 2, wherein the fluorescent group labeled by the probe for detecting bovine viral diarrhea virus is HEX, and the fluorescence quenching group is BHQ 1; the fluorescent group marked by the probe for detecting the bovine rotavirus is ROX, and the fluorescence quenching group is BHQ 2; the fluorescent group marked by the probe for detecting the bovine coronavirus is CY5, and the fluorescence quenching group is BHQ 3.

4. Use of the multiplex real-time fluorescent quantitative PCR primer and probe combination according to any one of claims 1 to 3 for simultaneous detection of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus for non-diagnostic purposes.

5. Use of the multiplex real-time fluorescent quantitative PCR primer and probe combination according to any one of claims 1 to 3 for the preparation of a kit for simultaneous detection of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus.

6. A kit for the simultaneous detection of bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus comprising the multiplex real-time fluorescent quantitative PCR primer and probe combination of any one of claims 1-3.

7. A method for simultaneously detecting bovine viral diarrhea virus, bovine rotavirus and bovine coronavirus for non-diagnostic purposes, comprising: comprising the step of adding the primer and probe set of any one of claims 1 to 3 to a reaction system and performing multiplex real-time fluorescent quantitative PCR amplification.

8. The detection method according to claim 7, wherein the reaction system is as follows: 2 XT 5 Fast qPCR Mix 12.5. mu.L; the upstream primer and the downstream primer for detecting the bovine viral diarrhea virus are respectively 0.7 mu L and 1.0 mu L of the probe; the upstream primer and the downstream primer for detecting the bovine rotavirus are respectively 0.8 mu L and 0.9 mu L of the probe; 0.8 muL of upstream primer and 1.0 muL of downstream primer and probe for detecting the bovine coronavirus respectively; 2 mu L of template DNA; make up to 25 μ L with double distilled water.

9. The detection method according to claim 7, wherein the reaction procedure of the amplification is: 5min at 95 ℃; annealing at 95 ℃ for 10s and 60 ℃ for 30s for 42 cycles, and fluorescence signals were automatically collected at the end of each cycle.