Triple PCR detection primer group, kit and detection method for bovine diarrhea pathogenic bacteria

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to a bovine diarrhea pathogenic bacterium triple PCR detection primer group, a kit and a detection method. The invention discloses a triple PCR (Polymerase Chain Reaction) detection primer group for three bovine diarrhea pathogenic bacteria causing bovine viral diarrhea mucosal disease virus, bovine rotavirus and bovine cobavirus, the sequences of the primer group are shown as SEQ ID NO: 1-6, and the invention discloses a triple PCR detection kit containing the primer group for the bovine diarrhea pathogenic bacteria and a detection method. Compared with the prior art, the detection method has the characteristics of high detection efficiency, low cost, strong specificity, good repeatability and high detection coincidence rate, is suitable for clinical high-throughput rapid diagnosis, and provides guarantee for prevention and control of pathogenic bovine diarrhea and reduction of economic loss.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and particularly relates to a triple PCR detection primer set, a kit and a detection method for bovine diarrhea pathogenic bacteria. Background Art

[0002] Pathogenic bovine diarrhea is mainly caused by bacterial, viral and parasitic infections. With the continuous development of large-scale and intensive breeding, the prevention and control situation of pathogenic bovine diarrhea is becoming increasingly severe. It not only affects the health of cattle, but also causes the death of cattle in severe cases, seriously affecting the economic benefits of large-scale cattle farms. Among them, viral infections cause relatively serious economic losses. Bovine viral diarrhea mucosal disease virus (BVDV), bovine rotavirus (BRoV) and bovine kobuvirus (BKoV) are important viruses causing bovine diarrhea. Bovine viral diarrhea mucosal disease virus causes enteritis, respiratory tract infection and diarrhea in cattle, resulting in abortion and reduced fertility in adult cattle; bovine rotavirus causes acute diarrhea in calves at one month old, with a relatively high mortality rate; bovine kobuvirus causes diarrhea, vomiting and fever in cattle, and ileal lesions occur in calves infected with BKoV. In addition, BKoV is often co-infected with other viruses. In the initial stage of infection with the above three viruses, the symptoms are similar. Often due to the lack of differential diagnosis methods and untimely diagnosis, symptomatic treatment cannot be carried out for bovine diarrhea, resulting in serious economic losses. There is an urgent need to develop a detection method and kit that can quickly judge the pathogenic virus at the initial stage of the disease, quickly determine the pathogenic virus, carry out symptomatic treatment, and timely prevent and control the spread of such viruses.

[0003] At present, the diagnosis of bovine viral diarrhea mucosal disease virus, bovine rotavirus and bovine kobuvirus mainly relies on pathogen isolation and single PCR technology. However, pathogen isolation is cumbersome and time-consuming, requiring cell culture and multiple passages, and it is difficult to meet the needs of rapid clinical detection; while single PCR detection requires three independent reactions respectively, which is not only time-consuming and laborious, but also increases the reagent cost and pollution risk. Especially in the face of the current situation of multi-pathogen mixed infection of bovine diarrhea disease, the single detection efficiency is low. At present, no technical system that can simultaneously detect the above three viruses has been established. Multiplex PCR can simultaneously amplify multiple target DNA fragments in the same reaction system and is applied to complex samples that need to simultaneously detect multiple pathogens. However, since multiplex PCR is carried out in the same system, there may be competitive inhibition between primers, templates, and between primers and templates during multiplex amplification. The concentration of each substance in the reaction system and the amplification conditions directly affect the results, and there are relatively large technical bottlenecks. At present, no report on a multiplex PCR detection method for simultaneously detecting the above three pathogenic viruses has been seen. Therefore, it is of great significance to develop a highly efficient, sensitive and specific triple PCR detection kit and detection method for bovine diarrhea virus. Summary of the Invention

[0004] To solve the above technical problems, the present invention selects the conserved gene sequences of BVDV, BRoV and BKoV published in the NCBI database, designs specific detection primers for the above three viruses, further optimizes the primer usage concentration, optimizes the PCR annealing temperature, and establishes a triple PCR detection method with high detection efficiency, strong specificity, good repeatability, high detection sensitivity and high coincidence rate; develops a triple PCR detection kit that can simultaneously detect three viruses, namely BVDV, BRoV and BKoV.

[0005] On the one hand, the present invention provides a triple PCR detection primer set for bovine diarrhea pathogens, and the triple PCR detection primer set includes: BVDV-402F, BVDV-402R, RV-288F, RV-288R, OKOVF, OKOV R, wherein the sequence of BVDV-402F is as shown in SEQ ID NO:1, the sequence of BVDV-402R is as shown in SEQ ID NO:2, the sequence of RV-288F is as shown in SEQ IDNO:3, the sequence of RV-288R is as shown in SEQ ID NO:4, the sequence of OKOV F is as shown in SEQ ID NO:5, and the sequence of OKOV R is as shown in SEQ ID NO:6; the bovine diarrhea pathogens are bovine viral diarrhea mucosal disease virus, bovine rotavirus and bovine kobuvirus.

[0006] On the second hand, the present invention also provides a kit containing the triple PCR detection primer set for bovine diarrhea pathogens.

[0007] Furthermore, the kit further includes a positive control, and the positive control is prepared by equally mixing the positive plasmids pUC57-BVDV5UTR, pUC57-RV-Vp6, and pUC57-OKoV-3D containing the conserved region sequences of BVDV, BRoV and BKoV.

[0008] On the third hand, the present invention also provides a triple PCR detection method for bovine diarrhea pathogens, and this method comprises the following steps:

[0009] Step 1: Extract RNA from the sample and synthesize cDNA;

[0010] Step 2: Using the cDNA synthesized in Step 1 as a template, perform a PCR amplification reaction with the triple PCR detection primer set described in Claim 1 to obtain a PCR product;

[0011] Step 3: Perform agarose gel electrophoresis analysis on the PCR product obtained in Step 2.

[0012] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, the sample is bovine feces.

[0013] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, the final concentrations of BVDV-402F and BVDV-402R in the triple PCR detection primer set are equal, and the final concentrations of BVDV-402F and BVDV-402R are 1.25 - 10 μmol / L; the final concentrations of RV-288F and RV-288R are equal, and the final concentrations of RV-288F and RV-288R are 1.25 - 10 μmol / L; the final concentrations of OKOV F and OKOV R are equal, and the final concentrations of OKOV F and OKOV R are 2.5 - 10 μmol / L.

[0014] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, the final concentration of BVDV-402F and BVDV-402R is 10 μmol / L, the final concentration of RV-288F and RV-288R is 10 μmol / L, and the final concentration of OKOV F and OKOV R is 10 μmol / L.

[0015] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, in the triple PCR amplification reaction of bovine diarrhea pathogenic bacteria, the annealing temperature is 50.4 ～ 61.6 °C.

[0016] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, the annealing temperature is 58.7 °C.

[0017] Further, in the triple PCR detection method for bovine diarrhea pathogenic bacteria, the amplified product fragment length of the bovine viral diarrhea mucosal disease virus by the triple PCR detection method for bovine diarrhea pathogenic bacteria is 402 bp, the amplified product fragment length of the bovine rotavirus is 193 bp, and the amplified product fragment length of the bovine kobuvirus is 629 bp.

[0018] In addition, the present invention also provides a reagent containing the triple PCR detection primer set for bovine diarrhea pathogenic bacteria.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] By selecting the conserved gene sequences of BVDV, BRoV, and BKoV published in the NCBI database, the present invention designs specific detection primers for bovine viral diarrhea mucosal disease virus, bovine rotavirus, and bovine kobuvirus as bovine diarrhea pathogenic bacteria, and through PCR process optimization, establishes a triple PCR detection method that can simultaneously detect three viruses, namely bovine viral diarrhea mucosal disease virus, bovine rotavirus, and bovine kobuvirus.

[0021] The triple PCR detection method for bovine diarrhea virus established in the present invention can amplify the DNA targets of three viruses, namely BVDV, BRoV and BKoV, in the same reaction system. Compared with single PCR detection, it can greatly save the detection reagent cost and time cost. In addition, when detecting three viruses by single PCR, each sample needs to be detected three times, which increases the sample consumption and the risk of sample contamination. The triple PCR can detect three target genes in one reaction, greatly reducing the risk of sample contamination.

[0022] The triple PCR detection method established in the present invention has been verified to have strong specificity, good repeatability, high detection compliance rate, especially excellent detection sensitivity. The lowest detection concentration of the triple PCR for BVDV virus is 11.40 fg / μL, for BRoV virus is 7.03 fg / μL, and for BKoV virus is 7.20 fg / μL. The lowest detection concentrations all reach the fg level.

[0023] The triple PCR detection kit for bovine diarrhea pathogenic bacteria developed based on the triple PCR detection method established in the present invention shows the characteristics of high detection efficiency, high sensitivity, strong specificity and high detection compliance rate in the detection of clinical samples. It is suitable for high-throughput rapid diagnosis in clinics, providing guarantee for preventing and controlling bovine diarrhea and reducing economic losses. Description of the Drawings

[0024] Figure 1 It is the electrophoresis detection result diagram of single PCR and triple PCR amplification products. Among them, the M lane is DL2000 Marker; the 1st lane is the triple PCR amplification product; the 2nd lane is the BVDV amplification product of single PCR; the 3rd lane is the BRoV amplification product of single PCR; the 4th lane is the BKoV amplification product of single PCR; the 5th lane is the negative control ddH2O.

[0025] Figure 2 It is the electrophoresis detection result diagram of triple PCR amplification products at different annealing temperatures. Among them, the M lane is DL2000 Marker; the 1st lane is the triple PCR amplification product at an annealing temperature of 50.4 °C; the 2nd lane is the triple PCR amplification product at an annealing temperature of 51.2 °C; the 3rd lane is the triple PCR amplification product at an annealing temperature of 53.3 °C; the 4th lane is the triple PCR amplification product at an annealing temperature of 55 °C; the 5th lane is the triple PCR amplification product at an annealing temperature of 58.7 °C; the 6th lane is the triple PCR amplification product at an annealing temperature of 60.8 °C; the 7th lane is the triple PCR amplification product at an annealing temperature of 61.6 °C; the 8th lane is the negative control ddH2O.

[0026] Figure 3Electrophoresis detection results of triple PCR amplification products of primer compositions with different concentrations. Lane M is DL2000 Marker; Lane 1 is 10 / 10 μmol / L BVDV-402F / R + 10 / 10 μmol / L RV-288F / R + 10 / 10 μmol / L OVOK F / R; Lane 2 is 10 / 10 μmol / L BVDV-402F / R + 10 / 10 μmol / L RV-288F / R + 10 / 10 μmol / L OVOK F / R; Lane 4 is 10 / 10 μmol / L BVDV-402F / R + 10 / 10 μmol / L RV-288F / R + 10 / 10 μmol / L OVOK F / R; Lane 4 is 10 / 10 μmol / L BVDV-402F / R + 10 / 10 μmol / L RV-288F / R + 10 / 10 μmol / L OVOK F / R; Lane 5 is negative control ddH2O.

[0027] Figure 4 Results of triple PCR specificity detection. Lane M is DL2000 Marker; Lane 1 is the triple PCR amplification product using the mixture of BVDV, BRoV, and BKoV positive plasmids as the template; Lane 2 is the single PCR product of BVDV; Lane 3 is the single PCR product of BRoV; Lane 4 is the single PCR product of BKoV; Lane 5 is the triple PCR product using the Sapovirus positive plasmid as the template; Lane 6 is the triple PCR product using the Norovirus positive plasmid as the template; Lane 7 is the triple PCR product using the Enteroviruses positive plasmid as the template; Lane 8 is the triple PCR product using Escherichia coli as the template; Lane 9 is the triple PCR product using Clostridium perfringens as the template; Lane 10 is negative control ddH2O.

[0028] Figure 5Results of triple PCR sensitivity detection. Lane M is DL2000 Marker; lanes 1 - 9 are triple PCR amplification products using mixtures of positive plasmids of BVDV, BRoV, and BKoV at different concentrations as templates. Among them, lane 1 is 11.40 ng / μL BVDV + 7.03 ng / μL BRoV + 7.20 ng / μL BKoV; lane 2 is 1.140 ng / μL BVDV + 0.703 ng / μL BRoV + 0.720 ng / μL BKoV; lane 3 is 0.114 ng / μL BVDV + 0.0703 ng / μL BRoV + 0.0720 ng / μL BKoV; lane 4 is 11.40 pg / μL BVDV + 7.03 pg / μL BRoV + 7.20 pg / μL BKoV; lane 5 is 1.140 pg / μL BVDV + 0.703 pg / μL BRoV + 0.720 pg / μL BKoV; lane 6 is 0.114 pg / μL BVDV + 0.0703 pg / μL BRoV + 0.0720 pg / μL BKoV; lane 7 is 11.40 fg / μL BVDV + 7.03 fg / μL BRoV + 7.20 fg / μL BKoV; lane 8 is 1.140 fg / μL BVDV + 0.703 fg / μL BRoV + 0.720 fg / μL BKoV; lane 9 is 0.114 fg / μL BVDV + 0.0703 fg / μL BRoV + 0.0720 fg / μL BKoV; lane 10 is the negative control ddH2O.

[0029] Figure 6 Results of within - batch repeatability test of triple PCR. Lane M is DL2000 Marker; lanes 1 - 3 are 3 within - batch repeats of template 1; lanes 4 - 6 are 3 within - batch repeats of template 2; lanes 7 - 9 are 3 within - batch repeats of the negative control ddH2O.

[0030] Figure 7 Results of between - batch repeatability test of triple PCR. Lane M is DL2000 Marker; lanes 1 - 3 are 3 between - batch repeats of template 1; lanes 4 - 6 are 3 between - batch repeats of template 2; lanes 7 - 9 are 3 between - batch repeats of the negative control ddH2O.

[0031] Figure 8 Results of clinical sample detection. Lane M is DL2000 Marker; lanes 1 - 16 are the detection results of clinical samples 1 - 16; lane 17 is the detection result of the positive control; lane 18 is the detection result of the negative control. Specific implementation mode

[0032] Next, the technical solution of the present invention will be described in conjunction with embodiments. However, the present invention is not limited to the following embodiments.

[0033] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. However, the embodiments cited do not limit the present invention.

[0034] In the following embodiments, the experimental methods and detection methods are all conventional methods unless otherwise specified; the reagents and materials can be obtained in the market unless otherwise specified.

[0035] Example 1

[0036] This example is for the establishment of a triple PCR detection method for bovine diarrhea virus.

[0037] 1.1 Design and synthesis of detection primers

[0038] According to the conserved gene sequences of BVDV, BRoV, and BKoV published in the NCBI database, specific primers for detecting three diarrhea viruses were designed using Oligo7, and the primers were synthesized by Invitrogen (Shanghai) Trading Co., Ltd. The primer information is shown in Table 1.

[0039] Table 1 Primer information

[0040]

[0041]

[0042] 1.2 Preparation of plasmid standard products

[0043] Using the positive sample for clinical detection of BVDV as a template, the conserved sequence BVDV5UTR of BVDV was amplified with BVDV-402F and BVDV-402R as primer pairs; using the positive sample for clinical detection of BRoV as a template, the conserved sequence RV-Vp6 of BRoV was amplified with RV-288F and RV-288R as primer pairs; using the positive sample for clinical detection of BKoV as a template and OKOV F and OKOV R as primer pairs to amplify the conserved sequence OKoV-3D of BKoV. The above amplification products were respectively ligated with the vector pUC57 to construct pUC57-BVDV5UTR (the positive plasmid of BVDV), pUC57-RV-Vp6 (the positive plasmid of BRoV), and pUC57-OKoV-3D (the positive plasmid of BKoV). The above three positive plasmids were transformed into DH5α competent cells, and after enrichment culture, the recombinant plasmids were extracted using the SanPrep column plasmid DNA miniprep kit and stored at -20°C.

[0044] 1.3 Single PCR Detection Method and Triplex PCR Detection Method

[0045] 1.3.1 Single PCR Detection Method

[0046] Using the positive plasmids of BVDV, BRoV, and BKoV as templates respectively, with BVDV-402F / R, RV-288F / R, and OKOV F / R in Table 1 as primers, and using ddH2O as a template as a negative control at the same time, single PCR amplification of BVDV, BRoV, and BKoV was carried out.

[0047] The reaction system of single PCR is: 5 μL of 2×Taq Master Mix, 0.5 μL of template, 1 μL each of the upstream and downstream detection primers at 10 mol / L, and the rest is made up to 10 μL with ddH2O.

[0048] The single PCR program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and the three steps of denaturation to extension are cycled 35 times, final extension at 72°C for 10 min, and the PCR product is stored at 4°C.

[0049] 1.3.2 Triplex PCR Detection Method

[0050] Mix the positive plasmids of BVDV, BRoV, and BKoV according to a mass ratio of 1:1:1 to prepare the template for triplex PCR. Mix the detection primers of the three viruses in Table 1 according to a ratio of 1:1:1 and carry out triplex PCR amplification.

[0051] The reaction system of triplex PCR is: 5 μL of 2×Taq Master Mix, 0.5 μL of triplex PCR template, 1 μL of the upstream and downstream detection mixed primers at 10 mol / L, and the rest is made up to 10 μL with ddH2O.

[0052] The triplex PCR program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and the three steps of denaturation to extension are cycled 35 times, final extension at 72°C for 10 min, and the PCR product is stored at 4°C.

[0053] 1.3.3 Detection of Single PCR Products and Triplex PCR Products

[0054] The single PCR products obtained in 1.3.1 and the triplex PCR products obtained in 1.3.2 were verified by electrophoresis using 1.2% agarose gel, and the results are as Figure 1As shown, the target gene fragment was not detected in the negative control ddH2O, indicating that the detection system was effective. The corresponding target gene fragments were amplified by both the single PCR method and the triple PCR method. Among them, the gene fragment size of BVDV was 402bp, the gene fragment size of BKoV was 629bp, and the gene fragment size of BRoV was 193bp, all of which were consistent with the expected sizes. It indicated that the triple PCR detection method for BVDV, BRoV, and BKoV viruses was successfully established.

[0055] 1.4 Optimization of the triple PCR detection method

[0056] To further improve the detection sensitivity and specificity of the triple PCR, different annealing temperatures and primers of virus genes with different concentrations were further set for the triple PCR detection method established in 1.3.2 to explore a better triple PCR detection system.

[0057] 1.4.1 Optimization of the annealing temperature of the triple PCR

[0058] Taking 50.4°C, 51.2°C, 53.3°C, 55°C, 58.7°C, 60.8°C, and 61.6°C as the annealing temperatures, the annealing temperature of the triple PCR was optimized, and other detection conditions were the same as those of the triple PCR detection in 1.3.2. The triple PCR products were detected by electrophoresis using 1.2% agarose gel. The results were as Figure 2 shown. When the annealing temperature was 58.7°C, the three target bands were the brightest, indicating that 58.7°C was the optimal annealing temperature.

[0059] 1.4.2 Optimization of the primer concentration of the triple PCR

[0060] Different primer combinations of BVDV-402F / R, RV-288F / R, and OKOV F / R were set to optimize the triple PCR system. The final concentrations of the primers in the BVDV-402F / R, RV-288F / R, and OKOV F / R primer combinations were: 10 / 10 μmol / L, 10 / 10 μmol / L, 10 / 10 μmol / L; 5 / 5 μmol / L, 10 / 10 μmol / L, 5 / 5 μmol / L; 2.5 / 2.5 μmol / L, 2.5 / 2.5 μmol / L, 5 / 5 μmol / L; 1.25 / 1.25 μmol / L, 1.25 / 1.25 μmol / L, 2.5 / 2.5 μmol / L.

[0061] Using the mixed positive plasmid composed of the positive plasmids of BVDV, BRoV, and BKoV mixed in a mass ratio of 1:1:1 as the template for the triple PCR, and using the above different primer combinations as primers for the triple PCR.

[0062] The reaction system of triple PCR is as follows: 5 μL of 2×Taq Master Mix, 0.5 μL of triple PCR template, 1 μL of detection mixed primers, and the rest is made up to 10 μL with ddH2O.

[0063] The triple PCR program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58.7°C for 30 s, extension at 72°C for 1 min. The three steps of denaturation to extension are cycled 35 times, and final extension at 72°C for 10 min. The PCR products are stored at 4°C.

[0064] The triple PCR products were detected by electrophoresis using 1.2% agarose gel, and the results are as Figure 3 shown. It shows that for the primer combination with the final concentrations of BVDV-402F / R, RV-288F / R, and OKOV F / R in the primer combination being 10 / 10 μmol / L, 10 / 10 μmol / L, and 10 / 10 μmol / L respectively, the target gene band in the triple PCR detection result is the brightest. This indicates that this concentration of primer combination is the optimal primer combination.

[0065] 1.5 Performance evaluation of the triple PCR detection method

[0066] 1.5.1 Specificity evaluation

[0067] The single PCR detection method in 1.3.1 was used to perform single PCR with the positive plasmids of BVDV, BRoV, and BKoV as templates respectively.

[0068] A mixed positive plasmid formed by mixing the positive plasmids of BVDV, BRoV, and BKoV in a mass ratio of 1:1:1 was used as the template for triple PCR. The positive plasmids of Sapovirus, Norovirus, Enteroviruses, and the total DNA of Escherichia coli and Clostridium perfringens were used as templates, and ddH2O was used as the template for the negative control. A primer combination with the final concentrations of BVDV-402F / R, RV-288F / R, and OKOV F / R being 10 / 10 μmol / L, 10 / 10 μmol / L, and 10 / 10 μmol / L respectively was used as the primer for triple PCR.

[0069] The reaction system of triple PCR is as follows: 5 μL of 2×Taq Master Mix, 0.5 μL of triple PCR template, 1 μL of the primer combination of 10 / 10 μmol / L BVDV-402F / R + 10 / 10 μmol / L RV-288F / R + 10 / 10 μmol / L OKOV F / R, and the rest is made up to 10 μL with ddH2O.

[0070] The triple PCR procedure is as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58.7°C for 30 s, extension at 72°C for 1 min. The three steps of denaturation to extension are cycled 35 times, and final extension is performed at 72°C for 10 min. The PCR products are stored at 4°C.

[0071] The single PCR products and triple PCR products were detected by electrophoresis using 1.2% agarose gel. The results are as Figure 4 shown. The corresponding target gene fragments were amplified by single PCR and triple PCR of BVDV, Rotavirus, and Kobuvirus. Among them, the gene fragment size of BVDV was 402 bp, the gene fragment size of BKoV was 629 bp, and the gene fragment size of BRoV was 193 bp, all of which were consistent with the expected sizes. However, no bands were observed in the PCR products amplified using the DNA of Sapovirus, Norovirus, Enteroviruses positive plasmids, Escherichia coli, and Clostridium perfringens as templates, and no bands were amplified in the negative control ddH2O. This indicates that the established triple PCR method has good specificity.

[0072] 1.5.2 Sensitivity evaluation

[0073] The concentrations of the positive plasmids of BVDV, BRoV, and BKoV were measured respectively. A dilution solution obtained by 10-fold serial dilution of the positive plasmids of BVDV, BRoV, and BKoV mixed in equal volumes with ddH2O was used as the template. The triple PCR detection method described in 1.5.1 was used to detect the lowest detection concentration of the three positive plasmids of BVDV, BRoV, and BKoV by the triple PCR, and the sensitivity of the triple PCR was evaluated. The results are as Figure 5 shown. The lowest concentrations of BVDV, BRoV, and BKoV that the triple PCR method could detect were 11.40 fg / μL, 7.03 fg / μL, and 7.20 fg / μL respectively, indicating that the triple PCR method established in this example has high sensitivity.

[0074] 1.5.3 Repeatability evaluation

[0075] Two portions of mixed positive plasmids were prepared according to the mass ratio of pUC57 - BVDV5UT:pUC57 - RV - Vp6:pUC57 - OKoV - 3D = 1:1:1, designated as template 1 and template 2. Using ddH2O as the negative control, the triple PCR detection method described in 1.5.1 was used to detect template 1 and template 2 simultaneously. Each sample was repeated 3 times to evaluate the within - batch repeatability of the method; the triple PCR detection method described in 1.5.1 was used to detect template 1 and template 2 at different times respectively, and each sample was repeated 3 times to evaluate the between - batch repeatability of the method. The results of the within - batch repeatability of the triple PCR detection method are as Figure 6As shown, the results of the between-batch repeatability of the triple PCR detection method are as Figure 7 shown. In the within-batch and between-batch repeatability tests of Template 1 and Template 2, the expected target bands were amplified in all repetitions, and no non-specific bands appeared, indicating that the triple PCR method has good repeatability.

[0076] Example 2

[0077] This example is for the detection of clinical samples.

[0078] 2.1 Extraction and reverse transcription of fecal RNA

[0079] Add 1 mL of sterile PBS to a 1.5 mL EP tube containing fecal samples, mix well by shaking, and let stand at room temperature for 5 min. Take 200 μL of the supernatant after sample treatment and transfer it to a new RNA-specific EP tube, then add 1 mL of RNAiso Plus, mix well by vortexing, and let stand on an ice box for 10 min. Add 200 μL of chloroform, vortex for 15 s, then let stand on an ice box for 10 min, and centrifuge at 4°C, 12000×g for 15 min.

[0080] The liquid is divided into 3 layers. Pipette 400 μL of the supernatant into a new 1.5 mL RNA-specific EP tube, add an equal volume of 400 μL of isopropanol, invert to mix well, let stand on an ice box for 10 min, and centrifuge at 4°C, 12000×g for 15 min. Pour off the supernatant, add 1 mL of 75% ethanol (treated with DEPC water). Centrifuge at 4°C, 12000×g for 10 min, pour off the supernatant, use a pipette to aspirate the excess ethanol, open the lid and air dry for 5 min, add 10 μL of RNase-Free ddH2O, pipette to mix well to dissolve the RNA.

[0081] Reverse transcribe the extracted RNA according to the FastKing cDNA First Strand Synthesis Kit. Prepare the reaction system for removing gDNA: Add 2 μL of 5×gDNA Buffer and 8 μL of Total RNA to the same RNA-specific EP tube, mix thoroughly, centrifuge briefly, and incubate in a 42°C water bath for 3 min, then place on ice; Prepare the reverse transcription reaction system: Add 2 μL of 10×KingRT Buffer, 1 μL of FastKing RT Enzyme Mix, 2 μL of FQ-RT Primer Mix, and 5 μL of RNase-Free ddH2O, and mix well. Add the reverse transcription reaction system to the gDNA removal reaction system, mix thoroughly, incubate in a 42°C water bath for 15 min, incubate in a 95°C water bath for 3 min, then place on ice to obtain the cDNA detection sample, and store it at -20°C for subsequent detection.

[0082] 2.2 Detection of clinical samples

[0083] Using the cDNA test sample prepared as in 2.1 as a template, the single PCR method and the established triple PCR method were respectively used for detection, and the coincidence rate was calculated (coincidence rate = detection rate of triple PCR method / detection rate of single PCR method × 100%), and the clinical application effect of the established triple PCR method was evaluated.

[0084] Single virus detection rate = number of positive detections of single virus / total number of samples

[0085] Two-virus detection rate = number of positive detections of 2 viruses in the same sample / total number of samples

[0086] Three-virus detection rate = number of positive detections of 3 viruses in the same sample / total number of samples

[0087] The cDNA obtained by reverse transcription of 200 bovine fecal samples was detected using the established triple PCR method. At the same time, a mixed positive plasmid composed of three virus positive plasmids of BVDV, BRoV and BKoV in a mass ratio of 1:1:1 was used as a positive control, and ddH2O was used as a negative control. The electrophoresis results of the triple PCR products of some samples are as Figure 8 shown. The detection rates of different viruses and the detection coincidence rates of the triple PCR are shown in Table 2. The detection rate of BVDV is 35% (70 / 200), the detection rate of BRoV is 12% (24 / 200), the detection rate of BKoV is 24% (48 / 200), the detection rate of BVDV + BRoV is 2.5% (5 / 200), the detection rate of BVDV + BKoV is 3.5%, the detection rate of BRoV + BKoV is 1% (2 / 200), and the detection rate of BVDV + BRoV + BKoV is 6.5% (13 / 200). The detection rate of single diarrhea virus is 33% (66 / 200), the detection rate of two diarrhea viruses is 8% (16 / 200), the infection rate of three diarrhea viruses is 6.5% (13 / 200), and the mixed infection accounts for 30.52% (29 / 95) of the total positive rate. After calculation, the coincidence rate of triple PCR and single PCR is 94.5 (189 / 200).

[0088] Table 2 Detection results of two PCR detection methods for clinical samples

[0089]

[0090] The triple PCR established by the present invention can amplify the DNA targets of three viruses, namely BVDV, BRoV and BKoV, in the same reaction system. While single PCR can only detect the DNA target of one virus at a time, and detecting three viruses by single PCR requires three PCRs, which greatly increases the consumption of reagents and samples. It can be seen that the triple PCR can greatly save the detection reagent cost and time cost. In addition, multiple detections of a single sample increase the risk of sample contamination. The triple PCR can detect three target genes in one reaction, greatly reducing the risk of sample contamination. In addition, the triple PCR detection method established by the present invention has been verified to have strong specificity, good repeatability, high coincidence rate and extremely high detection sensitivity. The lowest detection concentration of the triple PCR for the BVDV virus is 11.40 fg / μL, for the BRoV virus is 7.03 fg / μL, and for the BKoV virus is 7.20 fg / μL. Compared with the prior art, the triple PCR detection method established by the present invention has extremely high detection sensitivity and is suitable for high-throughput rapid diagnosis, providing a guarantee for preventing and controlling calf diarrhea and reducing economic losses.

[0091] As described above, the basic principles, main features and advantages of the present invention are preferably described. The above embodiments and the description are only for describing the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A triple-PCR detection primer set for bovine diarrhea pathogen, characterized in that, The bovine diarrhea pathogenic bacteria are bovine viral diarrhea mucosal disease virus, bovine rotavirus and bovine Kobuvirus. The triple PCR detection primer set includes: BVDV-402F, BVDV-402R, RV-288F, RV-288R, OKOV F, OKOV R. Among them, the sequence of BVDV-402F is as shown in SEQ ID NO:1, the sequence of BVDV-402R is as shown in SEQ ID NO:2, the sequence of RV-288F is as shown in SEQ ID NO:3, the sequence of RV-288R is as shown in SEQ ID NO:4, the sequence of OKOV F is as shown in SEQ ID NO:5, and the sequence of OKOV R is as shown in SEQ ID NO:

6.

2. A kit containing the triple PCR detection primer set for bovine diarrhea pathogenic bacteria described in claim 1.

3. The kit according to claim 2, wherein The kit further includes a positive control, which is prepared by equally mixing the positive plasmids pUC57-BVDV5UTR, pUC57-RV-Vp6, and pUC57-OKoV-3D plasmids containing the conserved region sequences of BVDV, BRoV, and BKoV.

4. A triple PCR detection method for bovine diarrhea pathogenic bacteria, characterized in that, Comprising the following steps: Step 1: Extract RNA from the sample and synthesize cDNA. Step 2: Using the cDNA synthesized in Step 1 as a template, perform a PCR amplification reaction with the triple PCR detection primer set for bovine diarrhea pathogenic bacteria described in claim 1 to obtain a PCR product. Step 3: Perform agarose gel electrophoresis analysis on the PCR product obtained in Step 2 to judge the detection result.

5. The triple-PCR detection method for bovine diarrhea pathogen according to claim 4, characterized in that, The sample is bovine feces.

6. The triple-PCR detection method for bovine diarrhea pathogens according to claim 4, wherein In the triple PCR detection primer set, the final concentrations of BVDV-402F and BVDV-402R are equal, and the final concentrations of BVDV-402F and BVDV-402R are 1.25 - 10 μmol / L; the final concentrations of RV-288F and RV-288R are equal, and the final concentrations of RV-288F and RV-288R are 1.25 - 10 μmol / L; the final concentrations of OKOV F and OKOV R are equal, and the final concentrations of OKOV F and OKOV R are 2.5 - 10 μmol / L.

7. The triple-PCR detection method for bovine diarrhea pathogen according to claim 6, characterized in that, The final concentration of BVDV-402F and BVDV-402R is 10 μmol / L, the final concentration of RV-288F and RV-288R is 10 μmol / L, and the final concentration of OKOVF and OKOV R is 10 μmol / L.

8. The triple-PCR detection method for bovine diarrhea pathogen according to claim 4, wherein In the triple PCR amplification reaction of bovine diarrhea pathogenic bacteria, the annealing temperature is 50.4 - 61.6 °C.

9. The triple PCR detection method for bovine diarrhea pathogen according to claim 8, wherein The annealing temperature is 58.7 °C.

10. The triple PCR detection method for bovine diarrhea pathogen according to claim 4, characterized in that, The amplified product fragment length of the triple PCR detection method for bovine diarrhea pathogenic bacteria for bovine viral diarrhea mucosal disease virus is 402 bp, the amplified product fragment length for bovine rotavirus is 193 bp, and the amplified product fragment length for bovine Kobuvirus is 629 bp.

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