Primer probe combination, kit and method for detecting bovine viral diarrhea virus nucleic acid
By designing specific primer probe combinations and TaqMan MGB qPCR technology, the problem of type distinction in viral diarrhea virus detection was solved, and stable and effective detection of BVDV type I and II was achieved, cross-reaction was reduced, and detection accuracy was improved.
Patent Information
- Application Number
- CN202510715412.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-26
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to quickly and effectively detect and distinguish between bovine viral diarrhea virus (BVDV) type I and type II and subtypes, and cross-reactions are prone to occur.
A primer probe combination is designed, including specific forward primers, reverse primers and fluorescent probes, and the BVDV 5’-UTR gene sequence differences are used for detection, and combined with TaqMan MGB qPCR technology, specific and high sensitivity detection of BVDV is achieved.
The specific detection of BVDV is realized, which can stably and effectively distinguish BVDV type I and II and their subtypes, reduce the possibility of cross-reaction, and has good clinical applicability and detection accuracy.
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Figure CN120536636A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of detection of bovine viral diarrhea virus nucleic acid, and in particular to a primer-probe combination, a kit and a method for detecting bovine viral diarrhea virus nucleic acid. Background Art
[0002] Bovine viral diarrhea virus (BVDV) infection has become a global public health problem. Its prevalence and severity are increasing, posing a serious threat to animal health and public health security. BVDV is a single-stranded positive-strand RNA virus with a genome size of approximately 12.3 kb. It belongs to the Flaviviridae family and the Pestivirus genus in taxonomy. pro Based on genetic sequence differences, BVDV genotypes are divided into BVDV type I, BVDV type II, and their genotype subtypes. Existing studies have shown that BVDV not only infects cattle, but also pigs, sheep, camels, yaks, and other animals. BVDV's rapid spread, wide host range, high variability, and adaptability together constitute the core of its epidemic characteristics, making its control and management more difficult. In this context, a comprehensive understanding of BVDV genotypes, the distribution of genotype subtypes, BVDV variation, and epidemic trends is crucial for developing broader-spectrum, more targeted vaccines and formulating targeted prevention and control strategies. Therefore, establishing a stable and effective method for detecting BVDV is of paramount importance. Summary of the Invention
[0003] The purpose of this application is to provide a primer-probe combination, kit, and method for detecting bovine viral diarrhea virus nucleic acid. The primer-probe combination provided in this application can specifically detect BVDV infection and can stably and effectively detect different subtypes of BVDV type I and BVDV type II. The specific technical solution is as follows:
[0004] The first aspect of the present application provides a primer-probe combination for detecting bovine viral diarrhea virus nucleic acid, comprising a forward primer with a sequence as shown in SEQ ID NO: 1, a reverse primer with a sequence as shown in SEQ ID NO: 2, and a probe with a sequence as shown in SEQ ID NO: 3.
[0005] In some embodiments of the present application, the fluorescent reporter group labeled at the 5' end of the probe is FAM; and the fluorescent quencher group labeled at the 3' end of the probe is MGB.
[0006] In some embodiments of the present application, the bovine viral diarrhea virus includes bovine viral diarrhea virus type I and bovine viral diarrhea virus type II.
[0007] In some embodiments of the present application, the primer-probe combination is unable to amplify bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and bovine infectious rhinotracheitis virus.
[0008] The second aspect of the present application provides a kit for detecting bovine viral diarrhea virus nucleic acid, wherein the kit comprises the primer-probe combination described in the first aspect of the present application.
[0009] The third aspect of the present application provides the use of the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application in detecting bovine viral diarrhea virus nucleic acid.
[0010] The fourth aspect of the present application provides a method for qualitatively detecting bovine viral diarrhea virus nucleic acid, which comprises the steps of:
[0011] S11: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested;
[0012] S12: Using the cDNA of the sample to be tested as a template, perform a qPCR (fluorescence quantitative PCR) amplification reaction using the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application;
[0013] S13: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or
[0014] The amplified product is sequenced, and the sequencing result is compared with the 5'-UTR gene sequence of the bovine viral diarrhea virus. Based on the consistency of the sequences, it is determined whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II.
[0015] In some embodiments of the present application, the qPCR amplification system includes: 0.3-0.5 μmol / L forward primer; 0.3-0.5 μmol / L reverse primer; 0.3-0.4 μmol / L probe; 1-20 ng / μL template cDNA; 2×Pro Taq HS Probe Premix II 0.4-0.6 μL / L.
[0016] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; and 40-50 cycles.
[0017] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 53-63°C, 25-35 s; 70-74°C, 25-35 s; and 40-50 cycles.
[0018] The fifth aspect of the present application provides a method for quantitatively detecting bovine viral diarrhea virus nucleic acid, which comprises the steps of:
[0019] S21: Perform qPCR amplification reaction on the diluted standards containing different copy numbers of bovine viral diarrhea virus plasmids, and draw a standard curve between the logarithm of the copy number of different plasmid standards and the cycle number Ct value;
[0020] S22: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested;
[0021] S23: Using the cDNA of the sample to be tested as a template, performing a qPCR amplification reaction using the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application;
[0022] S24: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or
[0023] Sequencing the amplified product, comparing the sequencing result with the 5'-UTR gene sequence of the bovine viral diarrhea virus, and judging whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II based on the consistency of the sequences;
[0024] S25: According to the cycle number Ct value of the sample to be tested, the bovine viral diarrhea virus load in the sample to be tested is converted using the standard curve.
[0025] In some embodiments of the present application, the qPCR amplification system includes: 0.3-0.5 μmol / L forward primer; 0.3-0.5 μmol / L reverse primer; 0.3-0.4 μmol / L probe; 1-20 ng / μL template cDNA; 2×Pro Taq HS Probe Premix II 0.4-0.6 μL / L.
[0026] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; and 40-50 cycles.
[0027] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 53-63°C, 25-35 s; 70-74°C, 25-35 s; and 40-50 cycles.
[0028] Beneficial effects of this application:
[0029] The present application provides a primer-probe combination for detecting bovine viral diarrhea virus nucleic acid, which can specifically detect BVDV infection and effectively detect different subtypes of BVDV type I and BVDV type II. The present application also provides a method for qualitatively or quantitatively detecting bovine viral diarrhea virus nucleic acid (universal BVDV TaqMan MGB qPCR detection method). Through specificity, sensitivity and clinical sample test results, it can be seen that the method provided in the present application can specifically detect BVDV and can achieve stable and effective detection of different genotypes and subtypes of BVDV.
[0030] Of course, it is not necessary to achieve all the advantages described above at the same time when implementing any product or method of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.
[0032] Figure 1 The consensus sequences of different subtypes of BVDVⅠ and BVDVII and the sequence regions for primer and probe design are shown;
[0033] Figure 2 This is the standard curve obtained in Example 2 of the present application;
[0034] Figure 3 This is a graph showing the specificity experiment results in Example 3 of the present application;
[0035] Figure 4 This is a diagram showing the sensitivity experiment results in Example 4 of this application. DETAILED DESCRIPTION
[0036] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in this application. Obviously, the embodiments described are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.
[0037] The first aspect of the present application provides a primer-probe combination for detecting bovine viral diarrhea virus nucleic acid, comprising a forward primer with a sequence as shown in SEQ ID NO: 1, a reverse primer with a sequence as shown in SEQ ID NO: 2, and a probe with a sequence as shown in SEQ ID NO: 3.
[0038] In some embodiments of the present application, the fluorescent reporter group labeled at the 5' end of the probe is FAM; and the fluorescent quencher group labeled at the 3' end of the probe is MGB.
[0039] Based on the differences in the 5'-UTR gene sequences of bovine viral diarrhea virus, type I and type II genotyping and the conservation of its own gene sequence, this application uses the BVDV 5'-UTR gene as a target to design detection primers and MGB probes. By comparing the sequences of the 5'-UTR genes of different gene subtypes of BVDVI and BVDVII, the conserved regions are selected for the design of universal detection primers and probes and the modification of probe MGB ( Figure 1 The consensus sequence of different subtypes of BVDVI type I and BVDVII and the primer and probe design sequence regions are shown. The sequences of the designed primer and probe combinations are as follows:
[0040] The forward primer (BVDV-F) was: 5′-CGAAGGCCGAAAAGAGGCTA-3′ (SEQ ID NO: 1);
[0041] The reverse primer (BVDV-R) was: 5′-GTCGAACCAYTGACGACT-3′ (SEQ ID NO: 2);
[0042] The probe (BVDV-P) is: 5′-FAM-ATGCCCTTWGTAGGACTAGC-MGB-3′ (SEQ ID NO: 3);
[0043] The length of the amplified product is 123 bp, and the fluorescent group signal is FAM.
[0044] In some embodiments of the present application, the bovine viral diarrhea virus includes bovine viral diarrhea virus type I and bovine viral diarrhea virus type II.
[0045] In some embodiments of the present application, the primer-probe combination is unable to amplify bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and bovine infectious rhinotracheitis virus.
[0046] The second aspect of the present application provides a kit for detecting bovine viral diarrhea virus nucleic acid, wherein the kit comprises the primer-probe combination described in the first aspect of the present application.
[0047] The third aspect of the present application provides the use of the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application in detecting bovine viral diarrhea virus nucleic acid.
[0048] The fourth aspect of the present application provides a method for qualitatively detecting bovine viral diarrhea virus nucleic acid, which comprises the steps of:
[0049] S11: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested;
[0050] S12: Using the cDNA of the sample to be tested as a template, performing a qPCR amplification reaction using the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application;
[0051] S13: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or
[0052] The amplified product is sequenced, and the sequencing result is compared with the 5'-UTR gene sequence of the bovine viral diarrhea virus. Based on the consistency of the sequences, it is determined whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II.
[0053] The present application has no particular limitation on the specific method for extracting RNA from the sample to be tested, as long as the purpose of the invention of the present application can be achieved. For example, the operation can be performed according to the instructions of the total RNA extraction reagent (Trizol) of Biosharp Company.
[0054] In some embodiments of the present application, the qPCR amplification system includes: 0.3-0.5 μmol / L forward primer; 0.3-0.5 μmol / L reverse primer; 0.3-0.4 μmol / L probe; 1-20 ng / μL template cDNA; 2×Pro Taq HS Probe Premix II 0.4-0.6 μL / L.
[0055] The present application has no particular limitation on the volume of the qPCR amplification system, as long as the purpose of the invention of the present application can be achieved. For example, the volume of the qPCR amplification system is 20 μL.
[0056] The present application has no particular limitation on the added volumes of the forward primer, reverse primer and probe, as long as the purpose of the invention of the present application can be achieved. For example, the added volumes of the forward primer and reverse primer are 0.2-1.2 μL respectively, and the added volume of the probe is 0.2-0.8 μL.
[0057] The present application has no particular limitation on the volume of template cDNA added, as long as the purpose of the invention of the present application can be achieved. For example, the volume of template cDNA added is 0.8-1.2 μL.
[0058] When preparing the qPCR amplification system, ddH2O can be used to supplement the qPCR amplification system to the preset volume, as long as the concentration of each component is within the range of this application.
[0059] The 2×Pro Taq HS Probe Premix II described in this application is a conventional universal reagent for qPCR system, which is commercially available and is not limited in this application.
[0060] The method of sequencing the amplified product, comparing the sequencing result with the 5'-UTR gene sequence of bovine viral diarrhea virus, and judging whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II based on the consistency of the sequence as described in the present application refers to comparing the sequencing result of the amplified product with the 5'-UTR gene sequences of bovine viral diarrhea virus type I and bovine viral diarrhea virus type II, respectively. If the sequencing result is the same as the 5'-UTR gene sequence of bovine viral diarrhea virus type I, the sample to be tested contains bovine viral diarrhea virus type I; if the sequencing result is the same as the 5'-UTR gene sequence of bovine viral diarrhea virus type II, the sample to be tested contains bovine viral diarrhea virus type II.
[0061] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; and 40-50 cycles.
[0062] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 53-63°C, 25-35 s; 70-74°C, 25-35 s; and 40-50 cycles.
[0063] This application has optimized the reaction system and reaction procedure of qPCR by matrix method. The study found that for the detection method of this application, the total system is 20 μL, the forward primer / reverse primer (BVDV-F / R) is 0.8 μL (10 μM) each, the probe (BVDV-P) is 0.6 μL (10 μM), and the two-step reaction procedure (i.e., 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; 40-50 cycles) is the optimal reaction condition, which can achieve more effective detection of BVDV.
[0064] The fifth aspect of the present application provides a method for quantitatively detecting bovine viral diarrhea virus nucleic acid, which comprises the steps of:
[0065] S21: Perform qPCR amplification reaction on the diluted standards containing different copy numbers of bovine viral diarrhea virus plasmids, and draw a standard curve between the logarithm of the copy number of different plasmid standards and the cycle number Ct value;
[0066] S22: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested;
[0067] S23: Using the cDNA of the sample to be tested as a template, performing a qPCR amplification reaction using the primer-probe combination described in the first aspect of the present application or the kit described in the second aspect of the present application;
[0068] S24: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or
[0069] Sequencing the amplified product, comparing the sequencing result with the 5'-UTR gene sequence of the bovine viral diarrhea virus, and judging whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II based on the consistency of the sequences;
[0070] S25: According to the cycle number Ct value of the sample to be tested, the bovine viral diarrhea virus load in the sample to be tested is converted using the standard curve.
[0071] This application achieves the purpose of quantitative analysis of the viral load of unknown samples by plotting the logarithm of the copy number of different plasmid standards versus the cycle number Ct value. For BVDV type I and BVDV type II, corresponding standard curves can be plotted separately.
[0072] When drawing the standard curve in the present application, for the doubly diluted standard containing bovine viral diarrhea virus plasmids with different copy numbers, a 10-fold dilution ratio can be used.
[0073] The present application has no particular limitation on the specific method for extracting RNA from the sample to be tested, as long as the purpose of the invention of the present application can be achieved. For example, the operation can be performed according to the instructions of the total RNA extraction reagent (Trizol) of Biosharp Company.
[0074] In some embodiments of the present application, the qPCR amplification system includes: 0.3-0.5 μmol / L forward primer; 0.3-0.5 μmol / L reverse primer; 0.3-0.4 μmol / L probe; 1-20 ng / μL template cDNA; 2×Pro Taq HS Probe Premix II 0.4-0.6 μL / L.
[0075] The present application has no particular limitation on the volume of the qPCR amplification system, as long as the purpose of the invention of the present application can be achieved. For example, the volume of the qPCR amplification system is 20 μL.
[0076] The present application has no particular limitation on the added volumes of the forward primer, reverse primer and probe, as long as the purpose of the invention of the present application can be achieved. For example, the added volumes of the forward primer and reverse primer are 0.2-1.2 μL respectively, and the added volume of the probe is 0.2-0.8 μL.
[0077] The present application has no particular limitation on the volume of template cDNA added, as long as the purpose of the invention of the present application can be achieved. For example, the volume of template cDNA added is 0.8-1.2 μL.
[0078] When preparing the qPCR amplification system, ddH2O can be used to supplement the qPCR amplification system to the preset volume, as long as the concentration of each component is within the range of this application.
[0079] The 2×Pro Taq HS Probe Premix II described in this application is a conventional universal reagent for qPCR system, which is commercially available and is not limited in this application.
[0080] The method of sequencing the amplified product, comparing the sequencing result with the 5'-UTR gene sequence of bovine viral diarrhea virus, and judging whether the sample to be tested includes bovine viral diarrhea virus type I and / or bovine viral diarrhea virus type II based on the sequence consistency as described in the present application refers to comparing the sequencing result of the amplified product with the 5'-UTR gene sequences of bovine viral diarrhea virus type I and bovine viral diarrhea virus type II, respectively. If the sequencing result is the same as the 5'-UTR gene sequence of bovine viral diarrhea virus type I, the sample to be tested contains bovine viral diarrhea virus type I; if the sequencing result is the same as the 5'-UTR gene sequence of bovine viral diarrhea virus type II, the sample to be tested contains bovine viral diarrhea virus type II.
[0081] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; and 40-50 cycles.
[0082] In some embodiments of the present application, the conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 53-63°C, 25-35 s; 70-74°C, 25-35 s; and 40-50 cycles.
[0083] This application uses the 5'-UTR gene sequence differences of bovine viral diarrhea virus (BVDV) to genotype I and II. Combining the conservation of the gene itself, detection primers were designed targeting the BVDV 5'-UTR gene. By comparing the 5'-UTR genes of different subtypes of BVDV type I and BVDV type II, and selecting their conserved regions for universal detection primer design, a universal BVDV TaqMan MGB qPCR detection method was established and initially applied to the detection of clinical samples, showing good clinical applicability.
[0084] The following examples are given to further illustrate the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.
[0085] Example 1
[0086] 1. RNA extraction and reverse transcription of test samples
[0087] RNA was extracted from the test sample according to the instructions of the total RNA extraction reagent (Trizol) of Biosharp Company to obtain RNA of the test sample (concentration was 382 ng / μL), and 2.6 μL of RNA was taken for reverse transcription into cDNA (concentration was 50 ng / μL).
[0088] 2. qPCR amplification of the sample to be tested
[0089] A 20 μL TaqMan MGB qPCR reaction system was established using 8 strips of qPCR tubes specifically for fluorescent quantitative PCR. The system consisted of 10 μL 2× Pro Taq HS Probe Premix II, 0.8 μL each of BVDV-F / BVDV-R (each at a concentration of 10 μM), 0.6 μL of BVDV-P (probe) (concentration of 10 μM), and 1 μL cDNA of the sample to be tested. ddH2O was added to 20 μL, mixed evenly, and then centrifuged.
[0090] Amplification conditions: a two-step method was used, 95°C for 2 min; 95°C for 5 s; 60°C for 30 s; and 45 cycles.
[0091] 3. Qualitative results analysis
[0092] After the reaction is complete, the amplification curve is analyzed and sequenced. The results show that the sample is positive, that is, an "S"-shaped amplification curve appears. The amplified product is sequenced and the amplified fragment is consistent with the target gene fragment.
[0093] Example 2
[0094] 1. Standard curve drawing
[0095] qPCR reaction was performed on 10-fold diluted standards containing different copy numbers of bovine viral diarrhea virus type I plasmid, and a standard curve was drawn between the logarithm of the copy number of different plasmid standards and the cycle number Ct value (such as Figure 2 As shown), specifically: Y = -3.355X + 41.56, correlation coefficient R 2 is 0.9977; where X is 1g (plasmid copy number) and Y is the Ct value.
[0096] The reaction system is as follows: 2×Pro Taq HS Probe PremixⅡ10μL, BVDV-F / BVDV-R 0.8μL each (concentration of each is 10μM), BVDV-P (probe) 0.6μL (concentration of 10μM), standard 1μL, ddH2O is added to 20μL, mixed evenly and centrifuged.
[0097] Amplification conditions: a two-step method was used, 95°C for 2 min; 95°C for 5 s; 60°C for 30 s; and 45 cycles.
[0098] 2. RNA extraction and reverse transcription of test samples
[0099] RNA was extracted from the test sample according to the instructions of the total RNA extraction reagent (Trizol) of Biosharp Company to obtain RNA of the test sample (concentration was 382 ng / μL), and 2.6 μL of RNA was taken for reverse transcription into cDNA (concentration was 50 ng / μL).
[0100] 3. qPCR amplification of the sample to be tested
[0101] A 20 μL TaqMan MGB qPCR reaction system was established using 8 strips of qPCR tubes specifically for fluorescent quantitative PCR. The system consisted of 10 μL 2× Pro Taq HS Probe Premix II, 0.8 μL each of BVDV-F / BVDV-R (each at a concentration of 10 μM), 0.6 μL of BVDV-P (probe) (concentration of 10 μM), and 1 μL cDNA of the sample to be tested. ddH2O was added to 20 μL, mixed evenly, and then centrifuged.
[0102] Amplification conditions: a two-step method was used, 95°C for 2 min; 95°C for 5 s; 60°C for 30 s; and 45 cycles.
[0103] 4. Qualitative results analysis
[0104] After the reaction, the amplification curve was analyzed and sequenced. The results showed that the sample was positive, with an "S"-shaped amplification curve. Sequencing of the amplified product confirmed that the amplified fragment was consistent with the target gene fragment of bovine viral diarrhea virus type 1.
[0105] 5. Quantitative results analysis
[0106] The cycle number Ct value of the sample to be tested is 25.77. According to Y=-3.355X+41.56, the load of bovine viral diarrhea virus type Ⅰ in the sample to be tested is converted to 5.086×10 4 copy.
[0107] Example 3
[0108] The established TaqMan MGB qPCR assay was used to detect clinical nucleic acid samples positive for BVDV type I, BVDV type II, bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and infectious bovine rhinotracheitis virus to evaluate its specificity.
[0109] 1. RNA extraction and reverse transcription of test samples
[0110] RNA was extracted and reverse transcribed into cDNA using the Biosharp Total RNA Extraction Reagent (Trizol) instructions for samples containing BVDV type I, BVDV type II, bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and infectious bovine rhinotracheitis virus. Three replicates were performed for each sample.
[0111] 2. qPCR amplification of the sample to be tested
[0112] Using 8 strips of qPCR tubes dedicated to fluorescent quantitative PCR, 20 μL of TaqMan MGB qPCR reaction system was established. The system was as follows: 10 μL of 2×Pro Taq HS Probe PremixⅡ, 0.8 μL of BVDV-F / BVDV-R (each at a concentration of 10 μM), 0.6 μL of BVDV-P (probe) (concentration of 10 μM), 1 μL of cDNA of the sample to be tested, and ddH2O was added to 20 μL. The mixture was mixed evenly and then centrifuged.
[0113] Amplification conditions: a two-step method was used, 95°C for 2 min; 95°C for 5 s; 60°C for 30 s; and 45 cycles.
[0114] 3. Results Analysis
[0115] After the reaction is completed, the amplification curve is analyzed and sequencing is performed for identification.
[0116] The results are as follows Figure 3 As shown, Figure 3 This is the specific experimental result diagram (amplification curve) in Example 3 of this application; among them, 1. BVDVⅠ; 2. BVDVⅡ; 3. Bovine coronavirus; 4. Bovine rotavirus; 5. Bovine parvovirus; 6. Bovine parainfluenza virus type 3; 7. Bovine infectious rhinotracheitis virus; 8. Negative control. Figure 3 As can be seen, only the test samples containing BVDV type I and BVDV type II showed amplification curves, while the test samples containing bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and bovine infectious rhinotracheitis virus did not show amplification curves. These results demonstrate that the universal TaqMan MGB qPCR detection method for BVDV provided in this application has strong specificity and does not cross-react with bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and bovine infectious rhinotracheitis virus.
[0117] Example 4
[0118] The copy numbers of BVDVⅠ plasmids were 1.265×10 4 , 1.265×10 3 , 1.265×10 2 , 1.265×10 1 , 1.265×10 0 Perform qPCR reactions with duplicated standard samples. Using eight qPCR tubes specifically designed for fluorescent quantitative PCR, set up a 20μL TaqMan MGB qPCR reaction system: 10μL 2× Pro Taq HS Probe Premix II, 0.8μL each of BVDV-F / BVDV-R (10μM each), 0.6μL of BVDV-P (probe) (10μM), 1μL of standard sample, and ddH2O to a final volume of 20μL. Mix thoroughly and centrifuge. Set up three replicates for each sample.
[0119] Amplification conditions: a two-step method was used, 95°C for 2 min; 95°C for 5 s; 60°C for 30 s; and 45 cycles.
[0120] After the reaction is completed, the amplification curve is analyzed. Figure 4 As shown, Figure 4 This is the sensitivity test result diagram (amplification curve) in Example 4 of the present application; where 1 is 1.265×10 4 Copy; 2 is 1.265×10 3 Copies; 3 is 1.265×10 2 Copies; 4 is 1.265×10 1 Copies; 5 is 1.265×10 0 6 copies; 6 is the negative control. Figure 4 As can be seen from the results, the universal TaqMan MGB qPCR detection method for BVDV established in this application can stably detect the target virus and can detect 1.265×10 0 Copy plasmid standards.
[0121] Example 5
[0122] 186 bovine serum samples were collected, of which 72 were from Lanzhou, Gansu Province, and 114 were from Northeast China. Each bovine serum sample was divided into two groups. One group was tested using the IDEXX BVDV nucleic acid detection kit (refer to the instructions for testing), and the other was tested using the universal BVDV TaqMan MGB qPCR detection method provided in this application: (1) RNA extraction and reverse transcription: referring to the instructions of the biosharp total RNA extraction reagent (Trizol), RNA was extracted from the bovine serum samples and reverse transcribed into cDNA; (2) qPCR amplification: using 8 rows of qPCR tubes dedicated to fluorescent quantitative PCR, 20 μL TaqMan MGB qPCR reaction systems were established respectively, the system was: 2×Pro Taq HS Probe PremixⅡ10μL, BVDV-F / BVDV-R 0.8μL each (concentration of each is 10μM), BVDV-P (probe) 0.6μL (concentration of 10μM), bovine serum sample cDNA 1μL, ddH2O is added to 20μL, mixed evenly and centrifuged; amplification conditions: use a two-step method, 95℃ 2min; 95℃ 5s; 60℃ 30s; 45 cycles.
[0123] The test results are shown in Table 1. As can be seen from Table 1, the overall positive rate of bovine serum samples was 36.02%. Among these, the positive rate of bovine serum samples from Lanzhou, Gansu Province was 51.39%, and the positive rate of bovine serum samples from Northeast China was 26.32%. Furthermore, the positive rate obtained using the universal BVDV TaqMan MGB qPCR detection method provided herein was 100% consistent with the positive rate obtained using the IDEXX BVDV nucleic acid detection kit.
[0124] Table 1 Test results of bovine serum samples
[0125] sample Number of positive samples Number of negative samples Positive rate Lanzhou area, Gansu Province 37 35 51.39％ Northeast China 30 84 26.32％ Total sample 67 119 36.02％
[0126] The amplified products of the positive bovine serum samples were sequenced and identified, and the results showed that the BVDV genotype of the bovine serum samples in Lanzhou, Gansu Province was type I, and the BVDV genotype of the bovine serum samples in Northeast China was type I.
[0127] The above-mentioned specificity, sensitivity and clinical sample test results indicate that the universal TaqMan MGB qPCR detection method provided in this application can specifically detect BVDV and can achieve stable and effective detection of different genotypes and subtypes of BVDV.
[0128] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.
Claims
1. A primer-probe combination for detecting bovine viral diarrhea virus nucleic acid, comprising a forward primer with a sequence as shown in SEQ ID NO: 1, a reverse primer with a sequence as shown in SEQ ID NO: 2, and a probe with a sequence as shown in SEQ ID NO:
3.
2. The primer-probe combination according to claim 1, wherein The fluorescent reporter group labeled at the 5' end of the probe is FAM; the fluorescent quencher group labeled at the 3' end of the probe is MGB.
3. The primer-probe combination according to claim 1, wherein The bovine viral diarrhea virus includes bovine viral diarrhea virus type I and bovine viral diarrhea virus type II.
4. The primer-probe combination according to claim 1 or 2, wherein The primer-probe combination cannot amplify bovine coronavirus, bovine rotavirus, bovine parvovirus, bovine parainfluenza virus type 3, and bovine infectious rhinotracheitis virus.
5. A kit for detecting bovine viral diarrhea virus nucleic acid, wherein: The kit comprises the primer-probe combination according to any one of claims 1 to 4.
6. Use of the primer-probe combination according to any one of claims 1 to 4 or the kit according to claim 5 in detecting bovine viral diarrhea virus nucleic acid.
7. A method for qualitatively detecting bovine viral diarrhea virus nucleic acid, wherein: Including steps: S11: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested; S12: Using the cDNA of the sample to be tested as a template, performing a qPCR amplification reaction using the primer-probe combination of any one of claims 1 to 4 or the kit of claim 5; S13: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or The amplified product is sequenced, and the sequencing result is compared with the 5'-UTR gene sequence of the bovine viral diarrhea virus. Based on the consistency of the sequences, it is determined whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II.
8. A method for quantitatively detecting bovine viral diarrhea virus nucleic acid, wherein: Including steps: S21: Perform qPCR amplification reaction on the diluted bovine viral diarrhea virus plasmid standards containing different copy numbers, and draw a standard curve between the logarithm of the copy number of different plasmid standards and the cycle number Ct value; S22: extracting RNA from the sample to be tested and performing reverse transcription to obtain cDNA of the sample to be tested; S23: Using the cDNA of the sample to be tested as a template, performing a qPCR amplification reaction using the primer-probe combination of any one of claims 1 to 4 or the kit of claim 5; S24: analyzing the amplification curve. If the amplification curve is S-shaped, the sample to be tested includes bovine viral diarrhea virus; and / or Sequencing the amplified product, comparing the sequencing result with the 5'-UTR gene sequence of the bovine viral diarrhea virus, and judging whether the sample to be tested includes bovine viral diarrhea virus type I or bovine viral diarrhea virus type II based on the consistency of the sequences; S25: According to the cycle number Ct value of the sample to be tested, the bovine viral diarrhea virus load in the sample to be tested is converted using the standard curve.
9. The method according to claim 7 or 8, wherein The qPCR amplification system includes: 0.3-0.5 μmol / L forward primer; 0.3-0.5 μmol / L reverse primer; 0.3-0.4 μmol / L probe; 1-20 ng / μL template cDNA; and 2×Pro Taq HS Probe Premix II 0.4-0.6 μL / L.
10. The method according to claim 7 or 8, wherein The conditions for qPCR amplification include: 94-96°C, 1-3 min; 94-96°C, 4-6 s; 58-62°C, 25-35 s; 40-50 cycles; or, 94-96°C, 1-3 min; 94-96°C, 4-6 s; 53-63°C, 25-35 s; 70-74°C, 25-35 s; 40-50 cycles.