Primer probe set and detection method for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus

Abstract

The present invention belongs to the technical field of virus detection, and is a primer probe group and detection method for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus. The PCR reaction system constructed based on the primer probe group for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus has the advantages of high sensitivity, strong specificity, good stability and good repeatability when used for molecular biological detection of bovine respiratory syncytial virus. It can detect as low as 10 copies / μL, can be used for BRSD epidemiological detection, and can accurately quantify the infection degree of BRSV, laying the foundation for carrying out BRSV molecular epidemiology and pathogenic mechanism. The PCR reaction system established based on the primer probe group for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus detects BRSV, which is beneficial to improve the detection sensitivity and efficiency of BRSV.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and specifically relates to a primer probe set and a detection method for real-time fluorescent quantitative PCR detection of bovine respiratory syncytial virus. Background Art

[0002] Bovine respiratory syncytial virus (BRSV) is a single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae. It is the pathogen of bovine respiratory syncytial disease (BRSD) and one of the main viral pathogens of bovine respiratory disease complex (BRDC). BRSV is mainly transmitted through respiratory aerosols and body surface contact and is highly contagious.

[0003] BRSV can infect a variety of ruminants, of which cattle are the most susceptible. Animals may not show any clinical symptoms after infection, and they serve as a source of infection to excrete toxins for a long time; in the acute stage, dairy cows are mainly characterized by severe interstitial pneumonia, accompanied by clinical symptoms such as fever, difficulty and rapid breathing, and a significant decrease or even complete loss of milk production. In severe cases, pregnant cows may suffer miscarriage. Every year, the production efficiency of farms attacked by respiratory diseases is greatly reduced, resulting in animal deaths, increased breeding costs such as treatment and prevention, and a great impact on the development of my country's animal husbandry. Establishing a rapid and accurate detection method for BRSD is an important measure to effectively control the occurrence and prevalence of BRSD and reduce the losses caused by the disease.

[0004] The methods currently used for BRSD detection mainly include serological diagnosis, virus isolation and identification, molecular biological diagnosis (reverse transcription-polymerase chain reaction, loop-mediated isothermal amplification technology, etc.). However, these existing detection techniques are relatively cumbersome and can only qualitatively analyze whether the virus exists.

[0005] For example, loop-mediated isothermal amplification (LAMP) is a new type of molecular biological detection method. This reaction can identify multiple different sequences of target DNA with the participation of Bst DNA polymerase, deoxynucleotide triphosphates (dNTPs), magnesium sulfate, betaine and enzyme buffer solution and multiple primers (inner primers are about 40 to 42 bp in length, outer primers are about 17 to 20 bp in length, and loop forward primers and loop backward primers are about 20 bp in length). This reaction does not require DNA denaturation, annealing, extension and other processes, and can be carried out at 60°C to 65°C. However, loop-mediated isothermal amplification technology has the disadvantages of low sensitivity, complex primer design, easy contamination, and can only perform qualitative detection but not quantitative detection of samples.

[0006] Reverse transcription polymerase chain reaction (RT-PCR) technology is a detection method for specific amplification of viral target gene fragments. In this detection method, the viral RNA must first be reverse transcribed into cDNA before PCR detection. The reaction system undergoes a three-step temperature cycle, undergoing denaturation, primer hybridization, and extension, so that the number of DNA molecules increases exponentially. After amplification, the target band is detected by agarose gel electrophoresis and other methods. Reverse transcription polymerase chain reaction technology has the advantages of rapidity, strong specificity, and high sensitivity, but it can only perform qualitative detection on samples, not quantitative detection on samples.

[0007] Real-time quantitative polymerase chain reaction (qPCR) is a pathogen detection technology based on PCR technology combined with DNA fluorescent labeling technology. Through the amplification process of qPCR, the number of cDNA molecules in the reaction mixture increases, and then the fluorescence intensity increases. qPCR detection technology overcomes the drawbacks of quantitative detection and false positives in PCR detection technology, has the advantages of high sensitivity and low false positive rate, and is an important technical means for detecting pathogens.

[0008] qPCR detection technology is mainly divided into fluorescent dye method and TaqMan fluorescent probe method. In the former method, the SYBR Green I used is a non-specific DNA labeling dye that can specifically bind to the minor groove on the DNA double strand and emit a fluorescent signal under the irradiation of the excitation light source. The intensity of the fluorescent signal can represent the number of double-stranded DNA molecules. Since the SYBR Green I dye can bind to all double-stranded DNA, primer dimers, single-stranded secondary structures and erroneous amplification products can also cause DNA molecules to fluoresce, affecting the accuracy of the detection. Therefore, it is necessary to use a melting curve to analyze the test results. However, the SYBR Green I dye method in real-time fluorescence quantitative PCR detection has the disadvantages of poor specificity and prone to false positives.

[0009] The TaqMan probe is a single-stranded oligonucleotide, the 5' end of the nucleotide sequence is modified with a luminescent group, and the 3' end is modified with a quencher group. When the probe is not bound to the DNA sequence, the fluorescent signal of the fluorescent group is absorbed by the quencher group. When the nucleic acid sequence is denatured and extended, the probe binds to the cDNA sequence, the fluorescent group and the quencher group separate, and the fluorescent group emits a fluorescent signal. As the amplification reaction continues, the number of DNA molecules increases, and the intensity of the fluorescent signal emitted by the fluorescent group gradually increases. After detection by the fluorescence detector, the corresponding relationship between the cycle threshold and the fluorescence intensity during the cyclic reaction process can be recorded in the software. In addition, the TaqMan probe method is more sensitive than the SYBR Green I dye method, and can effectively avoid false positive results caused by primer dimers.

[0010] Real-time fluorescence quantitative PCR technology can make up for the shortcomings of the above detection methods, and has many advantages such as improved sensitivity, strong specificity, and rapid quantitative detection. Therefore, as a detection method with high sensitivity and strong specificity, real-time fluorescence quantitative PCR is easy to operate and can also perform real-time quantitative detection of samples. Compared with other detection methods, the one-step real-time fluorescence quantitative PCR technology has the advantages of short operation cycle, high sensitivity, and quantitative detection of viruses.

[0011] In the prior art, a Chinese patent document with publication number CN113684315A records a primer probe combination, a kit and an application for detecting bovine respiratory virus, which can quickly and accurately identify four major bovine respiratory viruses (BHV-1, BRSV, BPIV-3 and BVDV) based on multiple fluorescent PCR. Since multiple viruses need to be detected simultaneously, the detection sensitivity is limited, and after the detection is completed, it is necessary to further confirm which one or which viruses belong to, and the detection efficiency of BRSV is low. Summary of the invention

[0012] The present invention aims to provide a primer probe set and a detection method for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus, so as to solve the technical problems of low sensitivity and low efficiency in the detection of bovine respiratory syncytial virus in the prior art.

[0013] In order to solve the above technical problems, the first aspect of the present invention is to provide a primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus, comprising:

[0014] The upstream primer BRSV-MF, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0015] Downstream primer BRSV-MR, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0016] The nucleotide sequence of the probe BRSV-MP is shown in SEQ ID NO.3.

[0017] Preferably, the 5' end of the probe carries a fluorescent reporter group, and the 3' end carries a fluorescent quencher group.

[0018] Preferably, the fluorescent reporter group is FAM, and the fluorescent quencher group is BHQ1.

[0019] The second aspect of the present invention is to provide a detection method for detecting bovine respiratory syncytial virus by real-time fluorescence quantitative PCR comprising the above-mentioned primer probe set, comprising the following steps:

[0020] (1) Extracting RNA from the virus sample to be tested;

[0021] (2) Performing PCR amplification reaction on the target band in RNA using upstream primer BRSV-MF, downstream primer BRSV-MR, probe BRSV-MP and qRT-PCR amplification kit;

[0022] (3) Analyze the PCR amplification reaction products.

[0023] Preferably, in step (2), the reaction system components for the PCR amplification reaction are as follows: 2×One StepU + Mix is ​​10 μL, One Step U + Enzyme Mix was 1 μL, 10 μmol / L upstream primer BRSV-MF was 0.4 μL, 10 μmol / L downstream primer BRSV-MR was 0.4 μL, 10 μmol / L probe BRSV-MP was 0.4 μL, Nuclease-free Water was 5.8 μL, plasmid template was 2 μL, and the reaction system was 20 μL.

[0024] Preferably, the detection limit of the plasmid template is 10 copies / μL.

[0025] Preferably, the reaction conditions in step (2) are: pre-denaturation at 95°C for 30 s; 95°C for 10 s, 54°C for 30 s, for a total of 40 cycles.

[0026] Preferably, in step (3), the analysis of the PCR amplification reaction product includes qualitative analysis and quantitative analysis using a fluorescent quantitative PCR standard curve, wherein the fluorescent quantitative PCR standard curve is constructed as follows:

[0027] The extracted bovine respiratory syncytial nucleic acid RNA was used as a template, reverse transcribed into cDNA using a reverse transcription kit, and the target band was amplified using the upstream primer BRSV-MF, the downstream primer BRSV-MR and a PCR amplification kit. The expected size of the amplified fragment was 141 bp. The PCR product was purified and connected with pMD18-T to construct the plasmid standard pMD18-T-BRSV-M.

[0028] The pMD18-T-BRSV-M plasmid standard was diluted 10-fold in a gradient manner. 9 ~10 2 8 dilutions of plasmid were used as templates, and three replicates were set for each dilution, and ddH 2 O is the negative control of the system. The template is amplified under appropriate reaction conditions, and the fluorescent quantitative PCR standard curve is drawn according to the Ct value in the amplification reaction result and the corresponding template copy number.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The PCR reaction system constructed based on the primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus has the advantages of high sensitivity, good stability, strong specificity and good repeatability when used for bovine respiratory syncytial virus detection. It can detect as low as 10 copies / μL, can be used for BRSD epidemiological detection, and can accurately quantify the degree of BRSV infection, laying the foundation for the molecular epidemiology and pathogenic mechanism of BRSV.

[0031] 2. The PCR reaction system constructed based on the primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus performs a one-step detection of the BRSV virus, which is beneficial to improving the detection sensitivity and efficiency of bovine respiratory syncytial virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1This is a fluorescence quantitative PCR standard curve established in one embodiment of the present invention.

[0033] Figure 2 This is a diagram showing the sensitivity verification results of real-time fluorescence quantitative PCR detection in one embodiment of the present invention.

[0034] Figure 3 This is a diagram showing the specificity verification results of real-time fluorescence quantitative PCR detection in one embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus

[0037] The primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus in the embodiment of the present invention includes:

[0038] The upstream primer BRSV-MF has a nucleotide sequence as shown in SEQ ID NO.1; the downstream primer BRSV-MR has a nucleotide sequence as shown in SEQ ID NO.2; and the probe BRSV-MP has a nucleotide sequence as shown in SEQ ID NO.3.

[0039] The primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus is designed as follows:

[0040] Based on all BRSV genome sequences in the NCBI database, sequence differences were analyzed after alignment, and sequence conserved regions were selected to design specific primers and probes. The designed primer sequences are as follows:

[0041] Upstream primer BRSV-MF: 5′-GGCAAATATGGAGACATACGTGAA-3′ (SEQ ID NO. 1);

[0042] Downstream primer BRSV-MR: 5'-GCAGAGATGGAYGATTGGAAC-3' (SEQ ID NO. 2);

[0043] The target fragment amplified by this primer is 141 bp in size, and Y in the downstream primer is a degenerate base, representing C / T.

[0044] The probe sequence designed for detecting bovine respiratory syncytial virus is as follows:

[0045] BRSV-MP: 5'-FAM-CTCCATGAAGGATCRACHTACACAGC-BHQ1-3' (SEQ ID NO.3);

[0046] Among them, FAM is the 5'-end labeled fluorescent reporter group, and BHQ1 is the 3'-end labeled fluorescent quencher group.

[0047] In the probe, R is a degenerate base, representing A / G; and H is a degenerate base, representing A / T / C.

[0048] Establishment of Real-time Fluorescence Quantitative PCR Detection System for Bovine Respiratory Syncytial Virus

[0049] (1) Based on the upstream primer BRSV-MF and the downstream primer BRSV-MR, the plasmid standard was constructed using the pMD18-T vector. The specific steps are as follows:

[0050] The extracted bovine respiratory syncytial nucleic acid RNA was used as a template, and reverse transcribed into cDNA using a reverse transcription kit (Takara, 6210a). The target band was PCR amplified using the bovine respiratory syncytial virus upstream primer BRSV-MF: 5'-GGCAAATATGGAGACATACGTGAA-3', the bovine respiratory syncytial virus downstream primer BRSV-MR: 5'-GCAGAGATGGAYGATTGGAAC-3' and a PCR amplification kit (Vazyme, P211). The expected size of the amplified fragment was 141 bp. The PCR product was purified and connected to pMD18-T to construct the plasmid standard pMD18-T-BRSV-M.

[0051] (2) Optimization of primer probe concentration and annealing temperature

[0052] (2.1) Annealing temperature optimization

[0053] Take 10 7 The pMD18-T-BRSV-M standard plasmid with 100 copies / μL was used as a template, and the qRT-PCR kit (Vazyme, Q223) system was used. Eight temperatures from 47°C to 58°C were selected for qRT-PCR temperature gradient reaction to explore the optimal annealing temperature of the primer probe. Among them, the annealing temperature was 58°C, 57.4°C, 56.2°C, 54°C, 51.3°C, 49.2°C, 47.7°C, 47°C, etc. 8 different temperatures were used for 40 cycles. The optimized reaction conditions for PCR amplification were: 95°C 30s pre-denaturation; 95°C 10s, 54°C 30s, a total of 40 cycles.

[0054] (2.2) Optimization of primer and probe concentration

[0055] Take 10 7 The pMD18-T-BRSV-M standard plasmid with 10 copies / μL was used as a template, and the qRT-PCR kit (Vazyme, Q223) was used to optimize the primer and probe concentrations in the reaction system. The primer concentrations were designed to be 0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L, 0.5μmol / L, 0.6μmol / L, 0.7μmol / L, 0.8μmol / L, and 0.9μmol / L, and the probe concentrations were designed to be 0.05μmol / L, 0.1μmol / L, 0.15μmol / L, 0.2μmol / L, 0.25μmol / L, 0.3μmol / L, 0.35μmol / L, 0.4μmol / L, and 0.45μmol / L. The optimal primer and probe concentration was determined to be 0.2μmol / L by the chessboard method.

[0056] After optimizing the concentration of primers and probes, the reaction system is as follows: 20 μL system contains the following components:

[0057]

[0058]

[0059] (2.3) Establishment of the standard curve for BRSV TaqMan real-time fluorescence quantitative PCR detection method

[0060] The pMD18-T-BRSV-M plasmid standard was diluted 10-fold in a gradient manner. 9 ~10 2 The plasmid with 8 dilutions of copies / μL was used as template and amplified using primer probe set. Three replicates were set for each dilution and ddH 2 O is the negative control of the system. The template is amplified using the qRT-PCR reaction conditions optimized in steps (2.1) and (2.2). The standard curve is drawn using Excel (Microsoft Excel 2019) based on the Ct value in the qRT-PCR reaction results and the corresponding template copy number.

[0061] Fluorescence quantitative PCR standard curve Figure 1 As shown in the experimental results, the qRT-PCR amplification curve is "S" type. 9 ~10 2 The amplification peak graph decreased according to the template concentration gradient within the concentration range of 100 copies / μL. The standard curve drawn according to the amplification results of qRT-PCR and the number of template copies was y=-0.2998x+11.724, R2 =0.9974, slope k = -0.2998, indicating that the standard curve has a good linear relationship.

[0062] (2.4) Validation of the sensitivity of the BRSV TaqMan real-time fluorescence quantitative PCR detection method

[0063] The pMD18-T-BRSV-M plasmid was diluted 10-fold in series to select a concentration of 10 9 ~10 1 The sensitivity of the detection method was verified by using pMD18-T-BRSV-M standard plasmid with 9 different concentrations such as 10 copies / μL as template.

[0064] Verification results are as follows Figure 2 As shown, the reaction system still has good amplification at 10 copies / μL.

[0065] exist Figure 2 Middle: Curve 10 is ddH 2 O; the concentrations of pMD18-T-BRSV-M plasmids represented by curves 1-9 are: 10 9 copies / μL, 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 copies / μL.

[0066] (2.5) Validation of the specificity of the BRSV TaqMan real-time fluorescence quantitative PCR detection method

[0067] The nucleic acids of eight pathogens, including bovine infectious rhinotracheitis virus (IBRV), bovine mycoplasma, bovine coronavirus, bovine parvovirus, standard strain of Staphylococcus aureus, bovine viral diarrhea / mucosal disease virus (BVDV), bovine enterococci, and BRSV, were amplified using the primer probe set designed previously and the optimized amplification conditions.

[0068] like Figure 3 The results showed that the pMD18-T-BRSV-M standard plasmid had an amplification curve, ddH 2 O had no amplification curve, indicating that the reaction system was not contaminated; only the system using BRSV nucleic acid as the template had an amplification curve, while the detection results of the other seven common respiratory pathogens were all negative, indicating that the primer probe had good specificity.

[0069] exist Figure 3 In the middle, curve S: pMD18-T-BRSV-M standard plasmid; curve N: ddH 2 O; Curve 1: BRSV; Curve 2: IBRV; Curve 3: Mycoplasma bovis; Curve 4: Bovine coronavirus; Curve 5: Bovine parvovirus; Curve 6: Staphylococcus aureus standard strain; Curve 7: BVDV; Curve 8: Enterococcus bovis.

[0070] (2.6) Verification of the repeatability of the BRSV TaqMan real-time fluorescence quantitative PCR detection method

[0071] The pMD18-T-BRSV-M plasmid was diluted 10-fold in series. 7 copies / μL, 10 6 copies / μL, 10 5 The pMD18-T-BRSV-M plasmid standard with 30 copies / μL was used as a template to verify the repeatability of the detection method. The intra-group repeat test was to perform 3 repeated tests on each concentration of the positive plasmid sample. The inter-group repeatability test was to perform 3 tests at different times. The coefficient of variation was used to determine the repeatability of the detection method.

[0072] The test results are shown in the table below. The coefficient of variation of each group was less than 2%, indicating that the real-time fluorescence quantitative detection method established in this experiment has good repeatability.

[0073] Table 1 Results of intra-group and inter-group repeatability tests of real-time fluorescence quantitative PCR

[0074]

[0075] It can be seen from the above detection system that the kit comprising the above primer probe set can be used for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus.

[0076] The above kit can be used in real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus in non-disease diagnosis and treatment methods, and comprises the following steps:

[0077] (1) Extract the total RNA of the virus sample to be tested.

[0078] (2) Then, the target band in the RNA was amplified by PCR using the upstream primer BRSV-MF, the downstream primer BRSV-MR, the probe BRSV-MP and a qRT-PCR amplification kit (Vazyme, Q223).

[0079] The reaction system components for the PCR amplification reaction here are as follows: 2×One Step U+ Mix is ​​10 μL, OneStep U + Enzyme Mix was 1 μL, 10 μmol / L upstream primer BRSV-MF was 0.4 μL, 10 μmol / L downstream primer BRSV-MR was 0.4 μL, 10 μmol / L probe BRSV-MP was 0.4 μL, Nuclease-free Water was 5.8 μL, plasmid template was 2 μL, and the reaction system was 20 μL.

[0080] (3) Analyze the PCR amplification reaction products, including qualitative analysis and quantitative analysis using a fluorescent quantitative PCR standard curve.

[0081] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer probe set for real-time fluorescence quantitative PCR detection of bovine respiratory syncytial virus, characterized in that: include: Upstream primer BRSV-MF, the nucleotide sequence of which is shown in SEQ ID NO.1; Downstream primer BRSV-MR, the nucleotide sequence of which is shown in SEQ ID NO.2; The nucleotide sequence of the probe BRSV-MP is shown in SEQ ID NO.

3.

2. The primer probe set for real-time fluorescent quantitative PCR detection of bovine respiratory syncytial virus according to claim 1, characterized in that: The 5' end of the probe carries a fluorescent reporter group, and the 3' end carries a fluorescent quencher group.

3. The primer probe set for real-time fluorescent quantitative PCR detection of bovine respiratory syncytial virus according to claim 2, characterized in that: The fluorescent reporter group is FAM, and the fluorescent quencher group is BHQ1.

4. A method for detecting bovine respiratory syncytial virus by real-time fluorescence quantitative PCR for non-disease diagnosis and treatment purposes, characterized in that: The following steps are involved: (1) Extracting viral RNA from the sample to be tested; (2) performing a PCR amplification reaction on the target band in the RNA using the upstream primer BRSV-MF, downstream primer BRSV-MR, probe BRSV-MP, and qRT-PCR amplification kit described in claim 1; (3) Analyze the PCR amplification reaction products.

5. The method according to claim 4, characterized in that: The reaction conditions in step (2) are: pre-denaturation at 95°C for 30s; 95°C for 10s, 54°C for 30s, for a total of 40 cycles.

6. The method according to claim 4, wherein: In step (3), the analysis of the PCR amplification reaction product includes qualitative analysis and quantitative analysis using a fluorescence quantitative PCR standard curve, wherein the fluorescence quantitative PCR standard curve is constructed as follows: Extracted bovine respiratory syncytial RNA was used as a template and reverse transcribed into cDNA using a reverse transcription kit. The target band was amplified using the upstream primer BRSV-MF and the downstream primer BRSV-MR using a PCR amplification kit. The expected amplified fragment size was 141 bp. The PCR product was purified and ligated with pMD18-T to construct the plasmid standard pMD18-T-BRSV-M. The pMD18-T-BRSV-M plasmid standard was diluted 10-fold in series. 9 ~10 2 A plasmid with 8 dilutions (10 copies / μL) was used as a template. Three replicates were set for each dilution, and ddH2O was set as a negative control. The template was amplified under appropriate reaction conditions, and a fluorescent quantitative PCR standard curve was drawn based on the Ct value in the amplification reaction results and the corresponding template copy number.

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