Detection method of porcine reproductive and respiratory syndrome virus type 1 strain
By designing a highly specific and sensitive TaqMan real-time RT-PCR detection method, the specificity and sensitivity issues of porcine reproductive and respiratory syndrome virus type 1 strain in existing technologies have been resolved, achieving rapid and accurate detection results.
Patent Information
- Application Number
- CN202511453888.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-13
- Publication Date
- 2026-01-09
AI Technical Summary
Existing methods for detecting porcine reproductive and respiratory syndrome virus type 1 strains have poor specificity and low sensitivity, which increases the difficulty of clinical prevention and control.
A highly specific and sensitive TaqMan real-time RT-PCR detection method was designed, which uses a specialized primer and probe combination to achieve rapid and accurate detection through real-time RT-PCR reaction.
It achieves highly sensitive and specific detection of porcine reproductive and respiratory syndrome virus type 1 strain, reduces the risk of laboratory contamination, is suitable for clinical testing, and has a low detection limit and good repeatability.
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Figure CN121294733A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection technology, specifically relating to a method for detecting porcine reproductive and respiratory syndrome virus type 1 strain. Background Technology
[0002] Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Its main characteristics include reproductive disorders in sows such as abortion, premature birth, and mummified fetuses, as well as respiratory symptoms in pigs of all ages. In pig production, affected pigs develop a distinct bluish-purple cyanosis of the ears, commonly known as "blue ear disease." PRRSV is classified under the order Nematoviruses (Nucleoviruses). Nidovirales Arteritis Virology Department ( Arteriviridae ), β arteritis virus genus ( Betaarterivirus The genome is a single-stranded positive-sense RNA of about 15kb with an envelope.
[0003] PRRSV can be divided into Betaarterivirus Europensis (Also known as porcine reproductive and respiratory syndrome virus type 1, PRRSV-1, or PRRSV European type) and Betaarterivirus americense (Also known as porcine reproductive and respiratory syndrome virus type 2, PRRSV-2, or PRRSV Americas), its nucleotide homology is approximately 55%–70%. Based on genetic evolution analysis of the ORF5 gene sequence, PRRSV-1 is divided into four subtypes (subtype 1–4), of which subtype 1 is further subdivided into the Lelystad-like branch (Clade A) and 11 other branches (Clade BL).
[0004] Currently, PRRSV-2 is the predominant strain circulating in my country, but PRRSV-1 has shown a trend of increasing prevalence in recent years. PRRSV-1 was first detected in samples originating in 2006. Strains represented by the LV-like strain were introduced into China, independently evolving into the native Chinese PRRSV-1, which has become the dominant strain. Subsequently, the PRRSV-1 strain GZ11-G1, highly homologous to vaccine strains, was isolated from Chinese pig herds. Recombinant mutations among PRRSV strains, coupled with the overlapping use of attenuated live vaccines targeting different strains in pig farms, have resulted in a diversity of PRRSV strains circulating in my country. The emergence of new strains has exacerbated the difficulty of PRRSV control in pig production.
[0005] Because cross-protection between PRRSV-1 and PRRSV-2 strains is very low, and their pathogenicity and clinical harm differ, differential diagnosis is crucial for selecting clinical prevention and control strategies. Currently, clinical methods for detecting porcine reproductive and respiratory syndrome virus (PRRSV) type 1 strains have poor specificity and low sensitivity. Therefore, developing a TaqMan real-time quantitative RT-PCR detection kit for PRRSV type 1 strains is of great significance for the detection and diagnosis of PRRSV in my country. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides a method for detecting porcine reproductive and respiratory syndrome virus type 1 (PRRSV) strains. Based on the re-screening of a highly sensitive and specific primer and probe combination for a PRSV type 1 strain, a rapid and accurate method for detecting PRSV type 1 strains is established.
[0007] This invention first provides a TaqMan real-time quantitative RT-PCR primer pair and probe for detecting porcine reproductive and respiratory syndrome virus type 1 strain, the sequence information of which is as follows: Upstream primer PRRSV-1-F: 5′-ATACATTCTGGCCCCTGCC-3′ (SEQ ID NO:1) Downstream primer PRRSV-1-R: 5′-ACAGCTCGTTTGCCGCC-3′ (SEQ ID NO:2) Fluorescent probe PRRSV-1-Probe: 5′-VIC-CACTCTAGTACCAGGRCTTCGG-MGB-3′ (SEQ ID NO:3).
[0008] The present invention also provides an application of the primer pair and probe described herein for the preparation of a TaqMan real-time RT-PCR detection kit for detecting porcine reproductive and respiratory syndrome virus type 1 strain.
[0009] This invention also provides a TaqMan real-time RT-PCR detection kit containing the above-described primer and probe composition.
[0010] This invention provides a diagnostic method for porcine reproductive and respiratory syndrome virus type 1 strain, comprising the following steps: 1) Obtain the cDNA from the sample to be tested; 2) Detection is performed using the primer and probe combination described above, or the kit described above; 3) Diagnose the porcine reproductive and respiratory syndrome virus type 1 strain based on the test results.
[0011] Furthermore, The detection includes: The cDNA of the sample to be tested was subjected to a real-time quantitative RT-PCR reaction in the detection system; The detection system includes: 2×qPCR Master Mix 12.5 μL; 10 μmol / L forward and reverse primers 0.75 μL each; 10 μmol / L probe 1 μL; cDNA template 5 μL; RNase-Free ddH2O 5 μL.
[0012] Furthermore, the reaction conditions of the detection system include: Pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s; primer annealing and extension at 60℃ for 30 s; fluorescence signal acquisition; 40 cycles.
[0013] This invention provides a primer and probe combination specifically targeting porcine reproductive and respiratory syndrome virus (PRRSV) type 1 strain. It possesses high specificity and sensitivity, enabling accurate and rapid detection of PRRSV type 1 strains. Furthermore, it exhibits a low detection limit and excellent repeatability, with a minimum detectable limit of 8.5 copies / μL. 3 ~1×10 8 It exhibits good repeatability (coefficient of variation <2%) within the gradient range of copies / μL.
[0014] The primer and probe combination and corresponding detection method for porcine reproductive and respiratory syndrome virus type 1 provided by this invention are highly specific and sensitive. The fluorescent quantitative RT-PCR products can be detected without opening the cap, reducing the risk of laboratory contamination and making them more suitable for clinical testing. Attached Figure Description
[0015] Figure 1 This is a sequence alignment diagram of primers and probes for the highly conserved region of the PRRSV genotype 1 strain provided in this embodiment of the invention; where F is the upstream primer, Probe is the probe, and R is the downstream primer.
[0016] Figure 2 This is a diagram showing the optimization results of the fluorescent quantitative RT-PCR primers provided in the embodiments of the present invention.
[0017] Figure 3 This is a diagram showing the optimization results of the fluorescence quantitative RT-PCR probe provided in the embodiments of the present invention.
[0018] Figure 4 This is a graph showing the optimization results of annealing temperature for real-time RT-PCR provided in this embodiment of the invention.
[0019] Figure 5These are the fluorescence quantitative RT-PCR amplification curve and standard curve provided in the embodiments of the present invention; wherein the upper figure is the amplification curve and the lower figure is the standard curve.
[0020] Figure 6 This is a graph showing the results of a specificity test for real-time RT-PCR provided in an embodiment of the present invention.
[0021] Figure 7 This is a graph showing the results of a fluorescence quantitative RT-PCR sensitivity test provided in an embodiment of the present invention.
[0022] Figure 8 This is a graph showing the results of a standard RT-PCR sensitivity test for PRRSV provided in an embodiment of the present invention. Detailed Implementation
[0023] This invention analyzes the gene sequences of various strains of porcine reproductive and respiratory syndrome virus (PRRSV) type 1. Thirty-one representative PRSV type 1 strain gene sequences from different countries and years were downloaded from GenBank. Multiple sequence alignment analysis was performed using Geneious Primer, identifying highly conserved regions within the conserved M protein sequence of PRSV. Highly conserved sequences meeting the above criteria were selected using TaqMan probe design principles: highly conserved fragments rich in CG, length 25–32 bp, Tm value 65–70°, CG content 30–80%, avoiding more than four consecutive Gs, and the first base at the 5' end not being G; and primer design principles: conserved regions with randomly distributed bases, length 18–25 bp, Tm value 55–62°, CG content 40–60%, avoiding primer dimers and hairpin structures. Probe and primer sequences were optimized using Primer Premier 5.0, Oligo 7.0, and Primer-BLAST. The optimized probe and primer sequences were validated using Nucleotide BLAST at NCBI. These sequences were 100% conserved across 100 BLAST sequences (the BLAST limit is 100) of porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strains. This design yielded a primer and probe combination with high specificity and sensitivity. The primer and probe combination designed for this region exhibits a lower detection limit compared to other publicly available conserved regions.
[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0025] Example 1: Design of screening primer and probe combinations This embodiment provides primers and probes for detecting porcine reproductive and respiratory syndrome virus type 1 strain. The specific procedure is as follows: 1. Primer and probe design This invention studies and analyzes the gene sequences of various strains of porcine reproductive and respiratory syndrome virus (PRRSV) type 1. Thirty-one representative strains of PRSV genotype 1 from different countries and years were downloaded from GenBank. Multiple sequence alignment analysis was performed using Geneious, revealing a highly conserved region within the conserved M protein sequence of PRSV. Probe and primer sequences were optimized using Primer Premier 5.0, Oligo 7.0, and Primer-BLAST. The optimized probe and primer sequences were validated using Nucleotide BLAST at NCBI, showing 100% conservation in 100 PRSV genotype 1 gene sequences (BLAST limit of 100). This design yields a primer and probe combination with high specificity and sensitivity. Figure 1 ).
[0026] The probes and primers were synthesized by Shanghai Sangon Biotech Co., Ltd. and Beijing Qingke Biotechnology Co., Ltd., respectively. Both probes and primers were diluted to 10 μM and stored at -20°C until use. The specific sequences are shown in the table below.
[0027] Table 1: Primer and probe sequence information for amplifying porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strain
[0028] 2. Optimization of conditions for quantitative real-time RT-PCR method The template was the whole genome plasmid DNA of porcine reproductive and respiratory syndrome virus type 1 strain. 0.25 μL, 0.50 μL and 1.25 μL of upstream and downstream primers were taken respectively to optimize the primer concentration.
[0029] Next, take the optimized primer concentrations, and aspirate 0.25 μL, 0.50 μL, and 1.00 μL of each probe. The template is positive recombinant plasmid DNA of the whole genome of porcine reproductive and respiratory syndrome virus, and then optimize the probe concentration.
[0030] Based on the optimized primer and probe final concentration preparation system, annealing temperatures of 62°C, 60°C, 59°C, and 57°C were used to determine the optimal annealing temperature for the reaction.
[0031] In a 25 μL amplification system, the minimum Cq value and the highest fluorescence intensity were selected as the criteria. The results showed that the optimal upstream and downstream primer concentrations were both 0.25 μM, the optimal probe concentration was 0.40 μM, and the optimal annealing temperature was 59°C. The optimized curves are shown below. Figure 2 , Figure 3 and Figure 4 .
[0032] Quantitative RT-PCR amplification was performed using optimized primers, probes, and annealing temperatures. The amplification system consisted of: 12.5 μL of 2×qPCR Master Mix; 0.75 μL each of 10 μmol / L forward and reverse primers; 1 μL of 10 μmol / L probe; 5 μL of cDNA template; and 5 μL of RNase-free ddH2O. The total volume was 25 μL. The amplification program was as follows: pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s; and primer annealing extension at 60℃ for 30 s. Fluorescence was collected at the end of the cycle, and 40 cycles were performed.
[0033] 3. Establishment of the standard curve The selected concentration is 1×10 3 ~1×10 8 Amplification was performed using copies / μL of positive plasmid DNA as a template, with RNase-free ddH2O as a negative control. An optimized quantitative RT-PCR amplification system and procedure were used to obtain the amplification curve and standard curve for porcine reproductive and respiratory syndrome virus (see [link to standard curve]). Figure 5 The results showed that the slope of the standard curve parameter for porcine reproductive and respiratory syndrome virus (PRRSV) was 3.687; R 2 The value was 0.999; the amplification efficiency (E) was 86.7%, indicating that the detection method had a good linear relationship and the amplification efficiency met the requirements.
[0034] 4. Specificity test Nucleic acid was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) genotype 2, porcine circovirus 2, porcine epidemic diarrhea virus (PEDV), classical classical swine fever virus (CSV), and porcine pseudorabies virus (PRV). PRSV genotype 1 nucleic acid was used as a positive control, and RNase-free ddH2O was used as a negative control. The established quantitative real-time RT-PCR method for PRSV genotype 1 was used for detection, and the analytical specificity of the method was evaluated.
[0035] The established methodology was then used to simultaneously perform qPCR tests on 6 PRRSV-negative specific pathogen-free (SPF) pig farms and 20 negative samples that had been tested with imported reagents. The diagnostic specificity of the detection method was evaluated based on the experimental results.
[0036] The method established in this study was used to simultaneously amplify nucleic acids from different pathogens as templates, and VIC fluorescence signals were collected. Specificity analysis results are shown below. Figure 6 This method can independently detect positive samples for porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 without causing nonspecific amplification of other viruses. The specificity analysis results are shown in Table 2. The analytical specificity of this method is 100%, demonstrating its excellent analytical specificity.
[0037] Table 2: Specificity Detection Results of Porcine Reproductive and Respiratory Syndrome Virus Genotype 1 Strain Analysis
[0038] Analytical specificity (DSp) 100% = 6 / (6+0) × 100%.
[0039] Twenty negative samples collected from PRRSV-negative SPF pig farms, which had been previously tested with imported reagents, were subjected to qPCR testing. The results of the diagnostic specificity analysis are shown in Table 3. For the negative samples, the diagnostic specificity of this method was 100%, demonstrating that the method has good diagnostic specificity.
[0040] Table 3: Results of Diagnostic Specificity Detection of Porcine Reproductive and Respiratory Syndrome Virus Genotype 1 Strain
[0041] Diagnostic specificity (DSp) 100% = 20 / (20 + 0) × 100% 5. Sensitivity test A concentration of 8.5 × 10⁻⁶ was selected. 9 ~8.5×10 -1 Using copies / μL of PRRSV-1 positive plasmid DNA as a template, conventional RT-PCR and the above-mentioned quantitative real-time RT-PCR were performed to determine the lowest copy number that could be detected by the two detection methods.
[0042] The procedure for a standard RT-PCR test is as follows: The reaction system includes: 2×PCR Mix 12.5 μL; 1 μL each of 10 μmol / L forward and reverse primers; 2 μL cDNA template; 8.5 μL RNase-Free ddH2O.
[0043] The reaction conditions include: Pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 55℃ and 72℃ for 30 s, for 34 cycles.
[0044] The results are as follows Figure 7 and Figure 8 As shown: the lowest detection limit of porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strain by quantitative real-time RT-PCR reached 8.5 copies / μL, while the lowest detection limit of conventional RT-PCR was 8.5 × 10⁻⁶. 2 copies / μL.
[0045] This indicates that the method established in this invention has good sensitivity.
[0046] 6. Repeatability test Select a concentration of 10 7 -10 3 Five gradients of plasmid DNA (copies / μL) were amplified using the previously established quantitative real-time RT-PCR method for porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strain. Repeatability tests were performed, and the coefficient of variation (Ct) was calculated to detect the repeatability of the method.
[0047] The results are shown in the table below: In three repeatability tests, the coefficient of variation of the Cq value was less than 2%, indicating that the method has good repeatability and stability.
[0048] Table 4: Results of Repeatability Tests on Porcine Reproductive and Respiratory Syndrome Virus Genotype 1 Strain Sample copy number (copies / μL) Cq average (MN) Cq standard deviation (SD) Coefficient of variation (%) <![CDATA[10 7 ]]> 14.663 0.175 1.193 <![CDATA[10 6 ]]> 18.497 0.040 0.216 <![CDATA[10 5 ]]> 22.300 0.167 0.749 <![CDATA[10 4 ]]> 25.703 0.128 0.498 <![CDATA[10 3 ]]> 29.370 0.051 0.174 7. Testing of clinical samples The established quantitative real-time RT-PCR method for porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strain was used to detect clinical pig tissue and serum samples submitted from all over the country. The results were compared with those obtained by conventional RT-PCR.
[0049] Thirty known clinical pig tissue and serum samples from across the country were tested. The results are shown in the table below. The established qPCR method detected 16 positive samples and 14 negative samples, with a positive detection rate of 53.33%. The conventional PCR method detected 10 positive samples and 20 negative samples, with a positive detection rate of 33.33%. The former had a 20.00% higher positive detection rate than the latter. The detection rate of the qPCR method was significantly higher than that of conventional PCR.
[0050] Table 5: Results of Clinical Sample Detection of Porcine Reproductive and Respiratory Syndrome Virus Genotype 1 Strain method Total number of samples Number of positive samples negative sample number Positive detection rate qPCR 30 16 14 53.33% Standard PCR 30 10 20 33.33% 8. Comparison of the sensitivity of detection reagents Thirty known clinical pig tissue and serum samples from across the country were collected. These samples had been previously sequenced and contained the PRRSV-1 strain. The newly developed qPCR method and Thermo Fisher VetMAX were used for the analysis. TM The PRRSV 3.0 qPCR detection kit was used to test 30 samples and compare Cq values to verify the sensitivity of the newly developed method in this embodiment.
[0051] The results are shown in the table below. Sixteen positive samples were detected using the aforementioned established quantitative RT-PCR method for porcine reproductive and respiratory syndrome virus genotype 1 strain. Thermo Fisher VetMAX was used to detect these samples. TM The number of positive samples detected by the PRRSV 3.0 reagent was 17.
[0052] Table 6: Comparison of the sensitivity of quantitative RT-PCR reagents for porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 strains.
[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A primer pair and probe for TaqMan real-time RT-PCR detection of porcine reproductive and respiratory syndrome virus type 1 strain, characterized in that, The primer pair has the sequence of the upstream primer as SEQ ID NO:1, the sequence of the downstream primer as SEQ ID NO:2, and the sequence of the probe as SEQ ID NO:
3.
2. The primer pair and probe as described in claim 1, characterized in that, The probe's 5' end is marked with VIC, and its 3' end is marked with MGB.
3. The application of the primer pair and probe described in claim 1 in the preparation of a TaqMan real-time RT-PCR detection kit for detecting porcine reproductive and respiratory syndrome virus type 1 strain.
4. A TaqMan real-time RT-PCR detection kit, characterized in that, The detection kit contains the primer pair and probe as described in claim 1.
5. A diagnostic method for porcine reproductive and respiratory syndrome virus type 1 strain, characterized in that, The method includes the following steps: 1) Obtain the cDNA from the sample to be tested; 2) Detection is performed using the primer pair and probe described in claim 1, or the kit described in claim 4; 3) Diagnose the porcine reproductive and respiratory syndrome virus type 1 strain based on the test results.
6. The method as described in claim 5, characterized in that, The detection in 2) involves performing a real-time quantitative RT-PCR reaction on the cDNA of the sample to be tested in the detection system. The detection system includes: 2×qPCR Master Mix 12.5 μL; 10 μmol / L forward and reverse primers 0.75 μL each; 10 μmol / L probe 1 μL; cDNA template 5 μL; RNase-Free ddH2O 5 μL.
7. The method as described in claim 5, characterized in that, The reaction conditions of the detection system are as follows: Pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s; primer annealing and extension at 60℃ for 30 s; fluorescence collection; 40 cycles.