TaqMan fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) kit and application thereof

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a TaqMan fluorescent quantitative RT-PCR kit and application thereof, the TaqMan fluorescent quantitative RT-PCR kit comprises primers and probes for detecting C-PRRSV, HP-PRRSV and NL-PRRSV respectively; the nucleotide sequences of the primer and the probe for detecting the C-PRRSV are as shown in SEQ ID NO.1-3; the nucleotide sequences of the primer and the probe for detecting the HP-PRRSV are as shown in SEQ ID NO. 4-6; the nucleotide sequences of the primer and the probe for detecting the NL-PRRSV are as shown in SEQ ID NO. 7-9. According to the present invention, the three strains such as C-PRRSV, HP-PRRSV and NL-PRRSV of the porcine reproductive and respiratory syndrome can be simultaneously detected, the advantages of good specificity, high sensitivity and good repeatability are provided, and particularly, the detection lower limit can be substantially reduced, and the sensitivity can be improved.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, and in particular to a TaqMan fluorescent quantitative RT-PCR kit and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute and highly contagious viral infectious disease of reproductive disorders and respiratory diseases in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is one of the most common infectious diseases in large-scale pig farms in my country and even in the world.

[0003] PRRSV has 10 open reading frames (ORFs), including ORF1a , ORF1b , ORF2a、ORF2b , ORF3, ORF4, ORF5, ORF5a, ORF6 and ORF7 .in, ORF2-5 Encoding structural proteins: GP2a, E, GP3, GP4, GP5, GP5a; ORF6 Encoding matrix protein (M); ORF7 Encodes viral nucleocapsid protein (N); ORF1 The protein can be further hydrolyzed into 16 non-structural proteins, including Nsp1α, Nsp1β, Nsp2-6, Nsp 2N, Nsp2TF, Nsp7α, Nsp7β and Nsp8-12; among them, the Nsp2 gene encodes the largest non-structural protein and plays an important role in viral replication. There are differences in the length of the Nsp2 gene among different virus strains, which makes it one of the targets for identifying different strains.

[0004] The classical strain (C-PRRSV), highly pathogenic strain (HP-PRRSV) and NADC-30like strain (NL-PRRSV) of porcine reproductive and respiratory syndrome virus are widely circulated in my country; therefore, it is necessary to detect the classical strain (C-PRRSV), highly pathogenic strain (HP-PRRSV) and NADC-30like strain (NL-PRRSV).

[0005] At present, some PRRSV detection methods have been developed based on the PCR principle to improve the sensitivity and accuracy of detection. Existing PRRSV detection methods include one-step multiplex PCR, real-time fluorescence quantitative PCR, nested PCR and reverse transcriptase PCR.

[0006] In the research of PRRSV detection methods, molecular biological detection technology represented by fluorescent quantitative PCR has the advantages of high sensitivity, strong specificity and good timeliness, and is widely used. However, the existing methods still have shortcomings, such as: the detection method can distinguish HP-PRRSV and C-PRRSV, but cannot detect PRRSV-1 or HP-PRRSV and C-PRRSV co-infection. In addition, although the existing TaqMan fluorescent quantitative PCR detection has the advantage of high sensitivity, the detection limit is basically 10 2 Approximately 10 copies / μL. Summary of the invention

[0007] The purpose of the present invention is to provide a TaqMan fluorescent quantitative RT-PCR kit and its application, which can not only simultaneously detect three strains of porcine reproductive and respiratory syndrome virus C-PRRSV, HP-PRRSV and NL-PRRSV, but also has the advantages of good specificity, high sensitivity and good repeatability, especially can greatly reduce the detection limit and improve the sensitivity, the detection limit of the three strains C-PRRSV, HP-PRRSV and NL-PRRSV is within 10 1 Approximately 10 copies / μL.

[0008] The present invention is achieved through the following technical solutions: A TaqMan fluorescent quantitative RT-PCR kit, wherein the TaqMan fluorescent quantitative RT-PCR kit can simultaneously detect C-PRRSV, HP-PRRSV and NL-PRRSV in porcine reproductive and respiratory syndrome; The TaqMan fluorescent quantitative RT-PCR kit includes primers and probes for detecting C-PRRSV, HP-PRRSV and NL-PRRSV respectively; The nucleotide sequence of the upstream primer for detecting C-PRRSV is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer for detecting HP-PRRSV is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6; The nucleotide sequence of the upstream primer for detecting NL-PRRSV is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.9.

[0009] The present invention compares and analyzes the Nsp2 regions of three strains of porcine reproductive and respiratory syndrome virus, C-PRRSV, HP-PRRSV and NL-PRRSV, and designs specific primers and probes respectively; when the specific primers and probes of the kit of the present invention are used for TaqMan fluorescent quantitative RT-PCR detection, only specific fragments of three target genotypes (C-PRRSV, HP-PRRSV and NL-PRRSV) can be amplified, and the specificity is good; the detection limit can be greatly reduced and the sensitivity can be improved. The detection limit of the three strains of C-PRRSV, HP-PRRSV and NL-PRRSV is within 10 1 copies / μL; the coefficient of variation within and between groups was between 0.10% and 2%, with good repeatability.

[0010] In summary, the TaqMan fluorescent quantitative RT-PCR kit of the present invention can not only simultaneously detect three strains of porcine reproductive and respiratory syndrome virus C-PRRSV, HP-PRRSV and NL-PRRSV, but also has the advantages of good specificity, high sensitivity and good repeatability, especially can significantly reduce the detection limit and improve sensitivity.

[0011] In a specific implementation, the TaqMan fluorescent quantitative RT-PCR kit also includes a reaction solution, which includes a buffer (2x One Step RT-PCR Buffer) and a reverse transcriptase (TaKaRa Ex Taq HS).

[0012] In a specific implementation, the TaqMan fluorescent quantitative RT-PCR kit also includes a positive control standard, and the positive control standard includes a C-PRRSV plasmid standard, a HP-PRRSV plasmid standard and a NL-PRRSV plasmid standard.

[0013] Among them, the nucleotide sequence of the C-PRRSV plasmid standard is shown in SEQ ID NO.10; the nucleotide sequence of the HP-PRRSV plasmid standard is shown in SEQ ID NO.11; and the nucleotide sequence of the NL-PRRSV plasmid standard is shown in SEQ ID NO.12.

[0014] In the specific implementation, a negative control solution is also included, and the negative control solution is RNase Free H 2 O).

[0015] The invention discloses an application of a TaqMan fluorescent quantitative RT-PCR kit in the detection of porcine reproductive and respiratory syndrome virus, wherein the detected strains include C-PRRSV, HP-PRRSV and NL-PRRSV.

[0016] When the above kit was used for TaqMan fluorescent quantitative RT-PCR detection, the RT-PCR reaction procedure was as follows: pre-denaturation at 95°C for 10 s; denaturation at 95°C for 10 s; annealing and extension at 55-58°C for 30 s, for 40 cycles.

[0017] In a preferred embodiment, the RT-PCR reaction procedure is: pre-denaturation at 95°C, 10 s; denaturation at 95°C, 10 s; annealing and extension at 57°C, 30 s, for 40 cycles.

[0018] When the above kit is used for TaqMan fluorescent quantitative RT-PCR detection, the RT-PCR reaction system is as follows: the concentration of the upstream primer and the downstream primer for detecting C-PRRSV are both 10 μmol / L, the volume is both 0.2~1.2µL, and the concentration of the probe for detecting C-PRRSV is 10 μmol / L, and the volume is 0.1~1µL; the concentration of the upstream primer and the downstream primer for detecting HP-PRRSV are both 10 μmol / L, the volume is both 0.2~1.2µL, and the concentration of the probe for detecting HP-PRRSV is 10 μmol / L, and the volume is 0.2~1.2µL; the concentration of the upstream primer and the downstream primer for detecting NL-PRRSV are both 10 μmol / L, the volume is both 0.1~0.6µL, and the concentration of the probe for detecting NL-PRRSV is 10 μmol / L, and the volume is 0.2~1.2µL.

[0019] In a preferred embodiment, the concentration of the upstream primer and the downstream primer for detecting C-PRRSV are both 10 μmol / L and the volume is 1.0 μL, and the concentration of the probe for detecting C-PRRSV is 10 μmol / L and the volume is 0.4 μL; the concentration of the upstream primer and the downstream primer for detecting HP-PRRSV are both 10 μmol / L and the volume is 0.6 μL, and the concentration of the probe for detecting HP-PRRSV is 10 μmol / L and the volume is 0.8 μL; the concentration of the upstream primer and the downstream primer for detecting NL-PRRSV are both 10 μmol / L and the volume is 0.2 μL, and the concentration of the probe for detecting NL-PRRSV is 10 μmol / L and the volume is 0.6 μL.

[0020] Based on the designed specific primers and probes, the present invention optimizes the concentration of the specific primers and probes in the RT-PCR reaction system and the RT-PCR reaction program, designs the RT-PCR reaction program that best matches the specific primers and probes in the kit, and can further reduce the detection limit of TaqMan fluorescent quantitative PCR, so that the detection limit of three strains of C-PRRSV, HP-PRRSV and NL-PRRSV is within 10 1 The detection limit of TaqMan fluorescence quantitative PCR is about 10 2 copies / μL, which greatly reduces the detection limit and greatly improves the sensitivity of the kit for TaqMan fluorescent quantitative PCR detection. It can be used for trace analysis of porcine reproductive and respiratory syndrome with high accuracy.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention compares and analyzes the Nsp2 regions of three strains of porcine reproductive and respiratory syndrome virus, C-PRRSV, HP-PRRSV and NL-PRRSV, and designs specific primers and probes respectively. The TaqMan fluorescent quantitative RT-PCR detection method can only amplify specific fragments of the three target genotypes, and has good specificity. According to the designed specific primers and probes, the concentrations of the specific primers and probes in the RT-PCR reaction system are optimized, and the RT-PCR reaction procedure is optimized. The detection limits of C-PRRSV, HP-PRRSV and NL-PRRSV can be greatly reduced, and the detection sensitivity of the kit can be improved. The detection limits of C-PRRSV, HP-PRRSV and NL-PRRSV are 10.1, 11.1 and 9.1 copies / μL, respectively, and the coefficients of variation within and between groups are between 0.10% and 2%, and the repeatability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings: Figure 1 Figure 2 is a diagram of amplified products of C-PRRSV, HP-PRRSV and NL-PRRSV of the present invention; Figure 2 Amplification curves of C-PRRSV, HP-PRRSV and NL-PRRSV when the annealing temperature is optimized according to the present invention; Figure 3 Amplification curves of C-PRRSV, HP-PRRSV and NL-PRRSV when the specific primers and probes of the present invention are optimized; Figure 4 Amplification curves of C-PRRSV, HP-PRRSV and NL-PRRSV in the specific experiment of the present invention; Figure 5 It is the amplification curve diagram of C-PRRSV, HP-PRRSV and NL-PRRSV in the sensitivity experiment of the present invention; Figure 6 The standard curve of TaqMan fluorescent quantitative RT-PCR of C-PRRSV of the present invention; Figure 7 The standard curve of TaqMan fluorescence quantitative RT-PCR of HP-PRRSV of the present invention; Figure 8 This is a standard curve diagram of TaqMan fluorescent quantitative RT-PCR of NL-PRRSV of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than 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.

[0024] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to those of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.

[0025] Embodiment 1: Materials and methods for one-step triple TaqMan fluorescence quantitative RT-PCR detection of porcine reproductive and respiratory syndrome: 1.1 Test materials 1.1.1 Materials and reagents: RNA nucleic acid extraction and purification kit (magnetic bead method) (Vazyme, China); proteinase K (Vazyme, China); One Step PrimeScript™ RT-PCR Kit (TaKaRa Dalian, Japan); TBGreen® Premix Ex Taq TM (TaKaRa Dalian, Japan).

[0026] 1.1.2 Instruments and equipment: Real-time fluorescence quantitative PCR instrument (Thermo Fisher Scientific, USA); Vazyme fully automatic nucleic acid extraction instrument (Vazyme, China); NanoDrop ONEC ultra-micro nucleic acid protein instrument (Thermo Scientific, USA); pipette (Eppendor, Germany); vortex shaker (Thermo Fisher Scientific, USA); high-speed refrigerated centrifuge XR3 (Thermo Fisher Scientific, USA); biological safety cabinet (Thermo Scientific, USA); ultra-low temperature refrigerator (Haier, China); high-pressure sterilizer (Zhiwei, China); fully automatic gel image analysis system (Ultra-Violet Products, USA).

[0027] 1.1.3 Source of samples: Clinical samples were provided by Sichuan Provincial Animal Disease Control Center. Standard plasmids of porcine reproductive and respiratory syndrome (classical type, highly pathogenic type, NADC30-like type) were purchased from Sangon Biotech (Shanghai) Co., Ltd. African swine fever virus (ASFV), classical swinesever virus (CSFV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine circovirus-2 (PCV-2), porcine circovirus-3 (PCV-3), porcine parvovirus infection virus (PV), and porcine transmissible gastroenteritis virus (TGEV) positive plasmids or vaccines were provided by the Animal Inspection Laboratory of Chengdu Customs Technical Center.

[0028] 1.1.4 Primer design: The reference sequences of porcine reproductive and respiratory syndrome classical, highly pathogenic and NADC30-like genomes were downloaded from NCBI, and the gene sequences were compared using DNAMAN software. The differences in the Nsp2 gene sequences of the classical strain (C-PRRSV), highly pathogenic strain (HP-PRRSV) and NADC-30-like strain (NL-PRRSV) of porcine reproductive and respiratory syndrome were compared and analyzed. Specific primers and probes for C-PRRSV, HP-PRRSV and NL-PRRSV were designed using primer 6.0 and oligo7 software, respectively. The specific primers and probes were put into Primer-Blast in NCBI to verify the specificity, and the best specific primers and probes were selected for synthesis. The nucleotide sequences of the specific primers and probes are shown in Table 1: Table 1

[0029] Note: The accession number of C-PRRSV in GenBank is AY032626.1, the accession number of HP-PRRSV is EF112445.1, and the accession number of NL-PRRSV is JN654459.1.

[0030] 1.2 Methods 1.2.1 Viral RNA extraction: The collected clinical sample tissues and vaccine viruses were resuspended in phosphate-buffered saline (PBS, pH 7.2). The samples were vortexed for 20 seconds in a biosafety cabinet. After standing for ≥ 30 seconds, 200 µL of the sample was aspirated and placed in a lysis plate for viral lysis and separation. The detailed extraction steps were carried out according to the instructions of the nucleic acid extraction and purification kit. Finally, the nucleic acid product eluted with 100 TE buffer was stored at -20°C.

[0031] 1.2.2 Preparation and dilution of positive plasmid: Synthesize the target fragment sequences of the three pairs of primers described in Table 1 of 1.1.4. After amplification of the target gene, the fragments are recovered and purified. The amplified sequences are shown in Table 2. Insert the PUC57 vector and connect with the target fragments to construct a recombinant plasmid standard. Then PCR amplify and verify the sequencing results. The prepared positive plasmid standard was measured for plasmid concentration using an ultra-micro nucleic acid protein analyzer. According to the formula, the number of plasmid copies = (M×6.02×10 23 ×10 -9 ) / (n×660), where M is the plasmid DNA concentration, n is the length of the recombinant plasmid = T vector length + target fragment length, calculate the number of positive plasmid copies, and dilute the positive plasmid 10 times to 10 1 -10 11 The diluted positive plasmid standard was stored in a -80℃ refrigerator.

[0032] Table 2

[0033] 1.2.3 One-step triple TaqMan fluorescence quantitative RT-PCR method system establishment and optimization: C-PRRSV, HP-PRRSV and NL-PRRSV triple TaqMan fluorescence quantitative RT-PCR reactions were performed according to the instructions of the One Step PrimeScript™ RT-PCR Kit (Perfect Real Time). The reaction system and procedure were based on the system and procedure optimized by the single TaqMan fluorescence quantitative RT-PCR in the previous preliminary experiment. The procedure was: pre-denaturation at 95°C for 10 s; denaturation at 95°C for 10 s, annealing and extension at 58°C for 30 s, and fluorescence was collected for 40 cycles. On this basis, the reaction procedure and primer probe concentrations were further optimized for the three pairs of designed primers using the synthesized positive plasmid. The reaction conditions were optimized for annealing temperature (53, 55, 57, 59, 61, and 63°C), primer concentration (primer concentration was 10 μmol / L, and the amount used for C-PRRSV and HP-PRRSV was 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, and 1.2; the amount used for NL-PRRSV was 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6), and probe concentration was 10 μmol / L (the amount used for HP-PRRSV and NL-PRRSV was 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 μL; the amount used for C-PRRSV was 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μL), as shown in Table 3. Then, the optimal primer and probe concentrations of the three genotypes were cross-combined for reaction to determine the optimal reaction conditions for triple TaqMan fluorescent quantitative RT-PCR.

[0034] Table 3

[0035] 1.2.4 Annealing temperature optimization: Figure 2As shown in the figure; the optimization of the annealing temperature of triple TaqMan fluorescent quantitative RT-PCR for C-PRRSV, HP-PRRSV and NL-PRRSV showed that there were significant differences in the Ct values ​​between the six temperature gradients set in the range of 53~63 ℃. When the annealing temperature was 55 and 57 ℃, the Ct values ​​of the three genotypes of C-PRRSV, HP-PRRSV and NL-PRRSV were small; and then the fluorescence signal intensity was compared. When the annealing temperature was 57 ℃, the virus Ct values ​​of the three genotypes were small and the fluorescence signal was the highest. Comprehensive consideration resulted in the optimal temperature of 57 ℃. At this annealing temperature, the fluorescence signal was strong, the amplification curve was smooth, and the Ct value was low. The reaction program of the one-step triple TaqMan fluorescent quantitative RT-PCR method was: 42 ℃, 5 min; 95 ℃, 10 s; 95 ℃, 5 s; 57 ℃, 30 s, 40 cycles.

[0036] The specific Ct values ​​of the three genotype viruses at different temperatures in the range of 53~63 ℃ are shown in Table 4: Table 4

[0037] From Table 4, we can see that: When the annealing temperature was 57 ℃, all three genotype viruses had lower Ct values.

[0038] 1.2.5 Optimization of primer and probe concentrations: First, the specific primer and probe concentrations of the two genotypes of C-PRRSV and HP-PRRSV were fixed, and the two optimal pairs of primer and probe concentration combinations for NL-PRRSV were selected according to the Ct value of the NL-PRRSV amplification curve, amplification efficiency, and fluorescence signal intensity (10 μmol / L, 0.2 μL, 0.4 μL; 10 μmol / L, 0.2 μL, 0.6 μL). The same method was used to select the optimal two pairs of primer and probe concentration combinations for C-PRRSV and HP-PRRSV (C-PRRSV: 10 μmol / L, 1 μL, 0.4 μL; 10 μmol / L, 1 μL, 0.6 μL; HP-PRRSV: 10 μmol / L, 0.6 μL, 0.6 μL; 10 μmol / L, 0.6 μL, 0.8 μL). Then, the two pairs of primer probes with the best concentrations for each of the three genotypes were cross-combined and reacted to select the combination with the least effect on the Ct value and fluorescence signal intensity of the three genotypes. Figure 3 The optimal reaction system of the one-step triple TaqMan fluorescent quantitative RT-PCR method is shown in Table 5. The optimized reaction procedure can reduce the amount of probes used without affecting the Ct value and fluorescence signal intensity of the genotype: Table 5

[0039] Embodiment 2: Target gene amplification and identification results: The positive plasmid was used as the template for amplification, and 3 pairs of primers were used for PCR amplification. The PCR products were detected by electrophoresis using 2% agarose gel. The gel image analysis system showed the following Figure 1 The results showed that the amplified bands were clear and bright, without dragging and diffusion, and the amplified product was consistent with the size of the target fragment. The sequencing results were compared on NCBI, and the homology with PRRSV reached more than 98%, confirming that the designed primers were effective in amplifying the target fragment.

[0040] Embodiment 3: Specificity Verification: The one-step triple TaqMan fluorescent quantitative RT-PCR detection method established in Example 1 was used to detect the other 7 related viruses. The results are as follows: Figure 4 As shown, only C-PRRSV, HP-PRRSV, and NL-PRRSV had positive amplification curves, while the other seven viruses (ASFV, CSFV, FMDV, PRV, PCV2, TGEV, PEDV) and the negative control had no amplification curves, indicating that the established one-step triple TaqMan fluorescent quantitative RT-PCR method has good specificity.

[0041] Embodiment 4: Sensitivity and standard curve: C-PRRSV, HP-PRRSV, NL-PRRSV mixed positive plasmids (concentrations were 2.02×10 7 ~2.02x10 0 , 1.11×10 7 ~1.11×10 0 and 1.82×10 7 ~1.82×10 0 The optimized method in Example 1 was used to perform a one-step triple TaqMan fluorescent quantitative RT-PCR reaction. The results are shown in Figure 1. Figure 5 As shown, the lowest concentration of the three viruses that can be detected is 10 1 copies / μL. Then 10 1The mixed positive plasmids were diluted two-fold in a gradient (the concentrations of positive plasmids for C-PRRSV, HP-PRRSV, and NL-PRRSV were 20.2, 10.1, and 5.05 copies / µL; 11.1, 5.55, and 2.77 copies / µL; and 18.2, 9.1, and 4.55 copies / µL, respectively) for triple TaqMan fluorescence quantitative RT-PCR reaction until no stable curve amplification ( Figure 5 ). The results showed that in the one-step triple TaqMan fluorescent quantitative RT-PCR detection method, the detection limit of C-PRRSV positive plasmid was 10.1 copies / µL, the detection limit of HP-PRRSV positive plasmid was 11.1 copies / µL, and the detection limit of NL-PRRSV positive plasmid was 9.1 copies / µL. Below this concentration, there was no amplification curve and no relevant fluorescence signal could be collected. Draw the standard curve as shown in Figure 6-Figure 8 As shown, at a nucleic acid copy number of 10 7 ~10 2 In the range of copies / µL, the standard curves of the three viruses all showed good linear relationships. The linear determination coefficients of C-PRRSV, HP-PRRSV, and NL-PRRSV ( R 2 ) are 0.997, 0.998 and 0.996 respectively.

[0042] Embodiment 5: Repeatability verification: Take 10 6 , 10 5 , 10 4 The mixed positive plasmids of C-PRRSV, HP-PRRSV, and NL-PRRSV with 100 copies / μL were used as templates to perform intra-group and inter-group repeat tests. The test results are shown in Table 6: The standard deviation (SD) of the parallel data of each group was less than 0.4, with a good central trend, and the coefficient of variation (CV%) within and between groups was between 0.10% and 2.00%. It can be seen that the established one-step triple TaqMan fluorescence quantitative RT-PCR detection method has good repeatability and stability.

[0043] Table 6

[0044] Embodiment 6: Actual sample detection: The one-step triple TaqMan fluorescence quantitative RT-PCR detection method established in Example 1 was used to detect 35 samples obtained from the Sichuan Provincial Animal Disease Prevention and Control Center during April 2024. The test results showed that the detection rate of the one-step triple TaqMan fluorescence quantitative RT-PCR detection method was 42.85% in 35 samples, which was consistent with the test results of the standard method.

[0045] In summary, among all virus detection technologies, molecular biology technologies represented by fluorescent quantitative PCR have obvious advantages in terms of detection sensitivity, specificity or stability. In particular, TaqMan fluorescent quantitative PCR releases fluorescent signals through targeted binding of probes to templates, which enhances the specificity of the reaction and achieves quantitative detection of viral nucleic acid copy numbers. According to comparative studies, the detection limit of TaqMan fluorescent quantitative PCR can usually reach 10 2 The number of copies / μL is about 100 and 1000 times that of SYBR Greenl dye-based fluorescent quantitative PCR and ordinary PCR, respectively. However, TaqgMan fluorescent quantitative PCR also has the disadvantages of expensive reagents and consumables and high cost of a single test. If multiple pairs of specific primers and probes are added to the same reaction system to detect multiple pathogens in one test, the operation time can be greatly shortened and the consumption of polymerase can be reduced. In addition, reverse transcriptase is added to the TaqMan fluorescent quantitative PCR reaction system to achieve the integration of RNA reverse transcription and cDNA amplification; its advantage is that it can effectively simplify the sample addition procedure, reduce contamination, and shorten the detection time.

[0046] Based on this, the present invention established a one-step multiplex TaqMan fluorescent quantitative RT-PCR detection method for three different genotypes of porcine reproductive and respiratory syndrome virus. First, by comparing and analyzing the Nsp2 regions of three strains of porcine reproductive and respiratory syndrome virus C-PRRSV, HP-PRRSV and NL-PRRSV, specific primers and probes were designed respectively. The TaqMan fluorescent quantitative RT-PCR detection method can only amplify specific fragments of the three target genotypes with good specificity. Then, based on the designed specific primers and probes, the concentrations of the specific primers and probes in the RT-PCR reaction system and the RT-PCR reaction program were optimized to design the RT-PCR reaction program that best matches the specific primers and probes in the kit (the optimal reaction program is: 42°C, 5 min; 95°C, 10 s; 95°C, 5 s; 57°C, 30 s, 40 cycles; the optimal reaction system is shown in Table 4), which can further reduce the TaqMan The detection limit of fluorescent quantitative PCR was 10 1The detection limit of TaqMan fluorescence quantitative PCR is about 10 2 copies / μL, which greatly reduces the detection limit and greatly improves the sensitivity of the kit for TaqMan fluorescent quantitative PCR detection. It can be used for trace analysis of porcine reproductive and respiratory syndrome with high accuracy; it can be used for rapid detection, differential diagnosis and epidemiological investigation of three different genotypes of viruses in batch samples.

[0047] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A TaqMan fluorescent quantitative RT-PCR kit, characterized in that: The TaqMan fluorescent quantitative RT-PCR kit can simultaneously detect C-PRRSV, HP-PRRSV and NL-PRRSV in porcine reproductive and respiratory syndrome; The TaqMan fluorescent quantitative RT-PCR kit includes primers and probes for detecting C-PRRSV, HP-PRRSV and NL-PRRSV respectively; The nucleotide sequence of the upstream primer for detecting C-PRRSV is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer for detecting HP-PRRSV is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6; The nucleotide sequence of the upstream primer for detecting NL-PRRSV is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the nucleotide sequence of the probe is shown in SEQ ID NO.

9.

2. A TaqMan fluorescent quantitative RT-PCR kit according to claim 1, characterized in that: Also included is a reaction solution, which includes a buffer and a reverse transcriptase.

3. A TaqMan fluorescent quantitative RT-PCR kit according to claim 1, characterized in that: Positive control standards are also included, including C-PRRSV plasmid standards, HP-PRRSV plasmid standards, and NL-PRRSV plasmid standards.

4. A TaqMan fluorescent quantitative RT-PCR kit according to claim 3, characterized in that: The nucleotide sequence of the C-PRRSV plasmid standard is shown in SEQ ID NO.10; the nucleotide sequence of the HP-PRRSV plasmid standard is shown in SEQ ID NO.11; and the nucleotide sequence of the NL-PRRSV plasmid standard is shown in SEQ ID NO.

12.

5. The TaqMan fluorescent quantitative RT-PCR kit according to claim 1, characterized in that: A negative control solution is also included, and the negative control solution is RNase-free water.

6. Use of a TaqMan fluorescent quantitative RT-PCR kit according to any one of claims 1 to 5 in the detection of porcine reproductive and respiratory syndrome, characterized in that: The strains tested included C-PRRSV, HP-PRRSV and NL-PRRSV.

7. The use according to claim 6, characterized in that: The RT-PCR reaction program was as follows: pre-denaturation at 95 °C for 10 s; denaturation at 95 °C for 10 s; annealing and extension at 55-58 °C for 30 s, for 40 cycles.

8. The use according to claim 7, characterized in that: The RT-PCR reaction program was as follows: pre-denaturation at 95 °C for 10 s; denaturation at 95 °C for 10 s; annealing and extension at 57 °C for 30 s, for 40 cycles.

9. The use according to claim 6, characterized in that: The RT-PCR reaction system is as follows: the concentration of the upstream primer and downstream primer used to detect C-PRRSV is 10 μmol / L, the volume is 0.2~1.2µL, and the concentration of the probe used to detect C-PRRSV is 10 μmol / L, the volume is 0.1~1µL; the concentration of the upstream primer and downstream primer used to detect HP-PRRSV is 10 μmol / L, the volume is 0.2~1.2µL, and the concentration of the probe used to detect HP-PRRSV is 10 μmol / L, the volume is 0.2~1.2µL; the concentration of the upstream primer and downstream primer used to detect NL-PRRSV is 10 μmol / L, the volume is 0.1~0.6µL, and the concentration of the probe used to detect NL-PRRSV is 10 μmol / L, the volume is 0.2~1.2µL.

10. The use according to claim 6, characterized in that: The RT-PCR reaction system was as follows: the concentrations of the upstream primer and downstream primer for detecting C-PRRSV were both 10 μmol / L and the volume was 1.0µL, and the concentration of the probe for detecting C-PRRSV was 10 μmol / L and the volume was 0.4µL; the concentrations of the upstream primer and downstream primer for detecting HP-PRRSV were both 10 μmol / L and the volume was 0.6µL, and the concentration of the probe for detecting HP-PRRSV was 10 μmol / L and the volume was 0.8µL; the concentrations of the upstream primer and downstream primer for detecting NL-PRRSV were both 10 μmol / L and the volume was 0.2µL, and the concentration of the probe for detecting NL-PRRSV was 10 μmol / L and the volume was 0.6µL.

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