Primer and probe combinations and detection kit for NADC34-like PRRSV-2 whole-genome multi-target detection

Abstract

The application discloses a primer probe combination and a detection kit for NADC34-like PRRSV-2 whole genome multi-target detection. The whole genome multi-target detection can detect multiple targets at the same time, significantly improves the detection efficiency, reduces the single detection cost, and identifies whether the virus is recombined. The high specificity and high sensitivity of the technology have obvious advantages in market competition. Through the multiple detection covering the whole genome, the technology can complete the detection of multiple gene targets in one reaction, reduces the repeated use of reagents and equipment, and significantly reduces the detection cost. This will make the accurate and rapid detection method more economical and feasible, and help to promote the use in the pig industry.

Description

Technical Field

[0001] This invention belongs to the field of viral nucleic acid detection, specifically involving primer and probe combinations and detection kits for NADC34-like PRRSV-2 whole genome multi-target detection. Background Technology

[0002] In recent years, NADC34-like porcine reproductive and respiratory syndrome virus (NADC34-like PRRSV-2) has become a major circulating strain in the swine industry. Frequent recombination events in its genome have led to significant differences in pathogenicity among strains. Existing research indicates that NADC34-like strains often undergo complex recombination with native strains such as NADC30-like and JXA1-like, forming chimeric viruses. These recombination events significantly increase the risk of virus transmission and exacerbate the difficulty of prevention and control. Therefore, developing detection technologies that can rapidly identify NADC34-like recombinant strains is crucial for epidemic monitoring. Currently, most RT-qPCR methods (such as the TaqMan probe method based on the ORF7 or Nsp2 genes) are mainly used for general PRRSV detection or typing. Although these methods have high sensitivity and good specificity, their target sites are mostly conserved regions, making it difficult to effectively identify the recombination characteristics specific to NADC34-like strains. For example, some methods identify NADC34-like strains solely through ORF5 RFLP genotyping (1-7-4 pattern), but genomic phylogenetic analysis reveals that these strains may carry recombinant fragments from other lineages, leading to genotyping results that do not accurately reflect the actual recombination status. Current recombination detection primarily relies on next-generation sequencing (NGS) and bioinformatics analysis (such as RDP software). While these methods can comprehensively resolve recombination sites, they are time-consuming and costly, making them unsuitable for large-scale rapid clinical screening. Recent studies have developed the SYBR Green RT-qPCR method targeting NADC34-like strains; however, due to the highly variable nature of NADC34-like strains, this method may miss detections due to detecting only a single target mutation, and it cannot yet distinguish between recombinant and non-recombinant strains.

[0003] The first inventor of this invention, Chen Nanhua, has been dedicated to the research of PRRSV differential diagnosis methods since 2006. He has published several SCI papers related to PRRSV detection (Chen et al., 2009. Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome Virus in China by a duplex real-time RT-PCR. Journal of Virological Methods, 161(2), 192-198. DOI:10.1016 / j.jviromet.2009.06.007. Chen et al., 2019. Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses. Transboundary and Emerging Diseases, 66(6):2271-2278.DOI:10.1111 / tbed.13276.), and applied for and obtained several invention patents related to PRRSV fluorescence quantitative PCR identification detection (PDRSV classic strain and highly pathogenic variant strain dual real-time fluorescence RT-PCR identification detection method, ZL200910077704.8; Porcine reproductive and respiratory syndrome virus universal real-time fluorescence RT-PCR detection method and kit, ZL201110073381.2). With more than ten years of rich experience in primer and probe design, when designing primer and probe combinations, we have fully considered and avoided most of the potential factors that may interfere with multiplex identification detection. These factors include, but are not limited to, dimers that may be formed between primer and probe sequences, mutual interference between primer and probe combinations of different targets, and differences in annealing temperature. However, despite careful consideration and optimization during the design phase, many challenges were still encountered during the actual experiment. These included mutual inhibition between primers and probes, synergistic effects between primers and probes, and, more complexly, incompatibility between some primer-probe combinations and any other primer-probe combinations, resulting in no amplification signal. This phenomenon may be related to severe mutual interference between primers and probes.This interference may stem from factors such as high homology, complementarity, or steric hindrance between primer and probe sequences, which prevent the primers and probes from binding normally to the target template during the reaction, thus preventing the amplification reaction from starting. Summary of the Invention

[0004] Purpose of the invention: To address the limitations of traditional detection methods, such as missed detections due to the susceptibility to mutations at single or few target sites and the inability to identify whether a virus strain has undergone recombination, this invention, through innovative experimental design and system optimization, successfully developed a multiplex real-time quantitative PCR technology based on a whole-genome multi-target detection strategy. This technology is specifically designed for the precise detection of genomic characteristics of NADC34-like PRRSV-2 strains, providing multiple primer-probe combinations. By simultaneously detecting multiple conserved regions of the viral genome and combining optimized primer-probe combination design, the ability to identify recombinant strains is significantly improved.

[0005] The technical problem that this invention also aims to solve is to provide a highly sensitive and specific detection kit containing the aforementioned primer-probe combination, which provides important technical support for detecting the recombination and evolution dynamics of NADC34-like strains and formulating precise prevention and control strategies.

[0006] Technical Solution: To solve the above-mentioned technical problems, this invention provides a primer-probe combination covering the entire genome of NADC34-like PRRSV-2, wherein the primer-probe combination includes any one or more of the following combinations:

[0007] The upstream primer N34-130F shown in SEQ ID NO.1, the probe N34-130P shown in SEQ ID NO.2, and the downstream primer N34-130R shown in SEQ ID NO.3; this primer-probe combination is named No. 1;

[0008] The upstream primer N34-1160F shown in SEQ ID NO.4, the probe N34-1160P shown in SEQ ID NO.5, and the downstream primer N34-1160R shown in SEQ ID NO.6; this primer-probe combination is named No. 2;

[0009] The upstream primer N34-1750F shown in SEQ ID NO.7, the probe N34-1750P shown in SEQ ID NO.8, and the downstream primer N34-1750R shown in SEQ ID NO.9; this primer-probe combination is named No. 4;

[0010] The upstream primer N34-1970F shown in SEQ ID NO.10, the probe N34-1970P shown in SEQ ID NO.11, and the downstream primer N34-1970R shown in SEQ ID NO.12; this primer-probe combination is named No. 5;

[0011] The upstream primer N34-4760F shown in SEQ ID NO.13, the probe N34-4760P shown in SEQ ID NO.14, and the downstream primer N34-4760R shown in SEQ ID NO.15; this primer-probe combination is named No. 8;

[0012] The upstream primer N34-5460F shown in SEQ ID NO.16, the probe N34-5460P shown in SEQ ID NO.17, and the downstream primer N34-5460R shown in SEQ ID NO.18; this primer-probe combination is named No. 10;

[0013] The upstream primer N34-6080F shown in SEQ ID NO.19, the probe N34-6080P shown in SEQ ID NO.20, and the downstream primer N34-6080R shown in SEQ ID NO.21; this primer-probe combination is named No. 12;

[0014] The upstream primer N34-6610F shown in SEQ ID NO.22, the probe N34-6610P shown in SEQ ID NO.23, and the downstream primer N34-6610R shown in SEQ ID NO.24; this primer-probe combination is named No. 13;

[0015] The upstream primer N34-6700F shown in SEQ ID NO.25, the probe N34-6700P shown in SEQ ID NO.26, and the downstream primer N34-6700R shown in SEQ ID NO.27; this primer-probe combination is named No. 14;

[0016] The upstream primer N34-8090F shown in SEQ ID NO.28, the probe N34-8090P shown in SEQ ID NO.29, and the downstream primer N34-8090R shown in SEQ ID NO.30; this primer-probe combination is named No. 15;

[0017] The upstream primer N34-10260F shown in SEQ ID NO.31, the probe N34-10260P shown in SEQ ID NO.32, and the downstream primer N34-10260R shown in SEQ ID NO.33; this primer-probe combination is named No. 17;

[0018] The upstream primer N34-10270F shown in SEQ ID NO.34, the probe N34-10270P shown in SEQ ID NO.35, and the downstream primer N34-10270R shown in SEQ ID NO.36; this primer-probe combination is named No. 18;

[0019] The upstream primer N34-11360F shown in SEQ ID NO.37, the probe N34-11360P shown in SEQ ID NO.38, and the downstream primer N34-11360R shown in SEQ ID NO.39; this primer-probe combination is named No. 19;

[0020] The upstream primer N34-12550F shown in SEQ ID NO.40, the probe N34-12550P shown in SEQ ID NO.41, and the downstream primer N34-12550R shown in SEQ ID NO.42; this primer-probe combination is named No. 21;

[0021] The upstream primer N34-13300F shown in SEQ ID NO.43, the probe N34-13300P shown in SEQ ID NO.44, and the downstream primer N34-13300R shown in SEQ ID NO.45; this primer-probe combination is named No. 24;

[0022] The upstream primer N34-14390F is shown in SEQ ID NO.46, the probe N34-14390P is shown in SEQ ID NO.47, and the downstream primer N34-14390R is shown in SEQ ID NO.48; this primer-probe combination is named No. 26.

[0023] Preferably, the present invention also includes any combination of the 16 sets of primer-probe combinations numbered 1, 2, 4, 5, 8, 10, 12-15, 17-19, 21, 24, and 26.

[0024] When performing single detection, any of the above primer-probe combinations can be selected.

[0025] When performing dual detection, the present invention also includes any pairwise combinations of the 16 primer-probe combinations numbered 1, 2, 4, 5, 8, 10, 12-15, 17-19, 21, 24, and 26 in the dual detection. There are 120 possible combinations of any two primer-probe combinations.

[0026] Preferably, the pairwise combinations included in the dual detection of the present invention are as follows: Combination 1: primer-probe combination numbered 1 (N34-130) and 5 (N34-1970); Combination 2: primer-probe combination numbered 2 (N34-1160) and 4 (N34-1750); Combination 3: primer-probe combination numbered 8 (N34-4760) and 10 (N34-5460); Combination 4: primer-probe combination numbered 12 (N34-6080) and 13 (N34-6610). Primer-probe combinations: Combination 5: Primer-probe combinations numbered 14 (N34-6700) and 18 (N34-10270); Combination 6: Primer-probe combinations numbered 15 (N34-8090) and 17 (N34-10260); Combination 7: Primer-probe combinations numbered 19 (N34-11360) and 24 (N34-13300); Combination 8: Primer-probe combinations numbered 21 (N34-12550) and 26 (N34-14390).

[0027] The probe is labeled with any fluorescein, and the other probe must be labeled with a fluorescein that uses a different detection channel. The fluorescein is selected from any one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 fluoresceins.

[0028] Among them, one or more of the NADC34-like PRRSV-2-SDLY23-1742 strain, NADC34-like PRRSV-2-BJ23-2652 strain, and NADC34-like PRRSV-2-rBJ1805-2 strain.

[0029] The concentrations of both the primers and probes are 5–20 μM. Preferably, the concentrations of both the primers and probes are 10 μM.

[0030] The present invention also includes the application of the primer-probe combination in the preparation of a multiplex detection kit for the whole genome of NADC30-like PRRSV-2 strains.

[0031] The present invention also includes a multiplex detection kit capable of simultaneously detecting the entire genome of the NADC34-like PRRSV-2 strain, wherein the multiplex detection kit includes the primer-probe combination described above.

[0032] The multiplex detection kit also includes other detection reagents required for multiplex PCR.

[0033] The multiplex PCR includes real-time quantitative PCR.

[0034] The multiplex assay kit includes 2×Premix Ex Taq and RNase-free H2O.

[0035] The amplification program of the multiplex detection kit is as follows: 1 cycle at 95℃ for 30 seconds; 40 cycles at 95℃ for 5 seconds and 60℃ for 1 minute.

[0036] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention, through whole-genome multi-target detection, can effectively distinguish NADC34-like PRRSV-2 from other PRRSV strains, significantly improving detection accuracy and reducing misdiagnosis and missed diagnosis caused by viral mutations. This invention achieves multiplex detection through primer-probe combinations, enabling simultaneous detection of multiple gene targets in a single reaction, greatly improving detection efficiency while reducing detection costs, making large-scale detection more economical and feasible. Because it covers multiple key gene regions of the viral genome, it can effectively detect recombination in various genes of NADC34-like PRRSV-2, providing important data support for viral evolution research and control. In summary, this invention, through whole-genome multi-target detection and recombination analysis technology, overcomes the shortcomings of existing technologies, providing stronger technical support for the detection, genetic evolution analysis, and effective control of NADC34-like PRRSV-2, and has significant practical significance and application value. Specifically, it includes the following aspects:

[0037] 1. By designing specific primer pairs and probes targeting multiple key gene regions of the NADC34-like PRRSV-2 genome, it is possible to effectively distinguish NADC34-like PRRSV-2 from other lineages. Traditional single-target detection methods are prone to false negatives when faced with high-frequency viral mutations and recombination, while the multi-target strategy significantly improves detection accuracy by covering both conserved and variant regions, reducing aquaculture losses due to missed detections and misjudgments.

[0038] 2. Since NADC34-like PRRSV-2 often recombines with other lineages (such as NADC30-like or HP-PRRSV), this method can effectively distinguish between non-recombinant NADC34-like strains and recombinant strains by targeting multiple conserved regions of the whole genome, providing more accurate molecular typing basis for epidemiological investigations.

[0039] 3. Multiplex detection reduces reagent consumption and equipment reuse, lowering the cost per sample to half that of conventional methods. Compared to traditional RT-PCR or ordinary RT-qPCR, this method uses a premixed reaction system or TaqMan probe design, simplifying the operation process and shortening the detection time. It also avoids the non-specific amplification problem of the SYBR Green method, making it more suitable for large-scale clinical rapid testing and accurate analysis needs.

[0040] 4. Currently, the NADC34-like strain is prevalent in many regions, but the protective effect of existing commercial vaccines is limited. This invention can provide key technical support for the early monitoring, vaccine research and development evaluation, and precise prevention and control of this strain, reducing economic losses caused by false or missed detections.

[0041] 5. This invention enables simultaneous detection of multiple targets of NADC34-like PRRSV-2. The application of this technology has significant monitoring value for pig farms with PRRSV infection. Regular testing allows for real-time monitoring of changes in the genetic characteristics of the virus within the pig herd. If a change in the detected gene combination is found in a particular test, this usually indicates the possible introduction of a new strain into the herd. Timely detection of such changes provides crucial data for pig farms, enabling early implementation of control measures to prevent the widespread transmission of new strains and effectively reduce economic losses caused by disease. Attached Figure Description

[0042] Figure 1 This is an example diagram of a genome-wide alignment and screening design for NADC30-like PRRSV-2.

[0043] Figure 2A This is an example of the good specificity of the NADC34-like PRRSV2 whole-genome multi-target detection method.

[0044] Figure 2B This is an example of poor specificity in the NADC34-like PRRSV2 whole-genome multi-target detection method;

[0045] Figure 3A The figure shows the test results of the NADC34-like PRRSV2 whole-genome multi-target detection method for detecting non-recombinant strain (rBJ1805-2);

[0046] Figure 3B The graph shows the test results of the NADC34-like PRRSV2 whole-genome multi-target detection method for detecting recombinant strain (SDLY23-1742);

[0047] Figure 3CThis is a graph showing the recombination analysis results of strains rBJ1805-2 and SDLY23-1742;

[0048] Figure 4A This is an example of the inhibitory combination results of a genome-wide multi-target multiplex detection method for NADC34-like PRRSV-2;

[0049] Figure 4B This is an example of optimized combination results for NADC34-like PRRSV-2 with multiple targets and multiple detections across the entire genome;

[0050] Figure 5 This is a graph showing the test results of the NADC34-like PRRSV-2 whole-genome multi-target multiplex detection method V1 kit;

[0051] Figure 6 This is a graph showing the test results of the NADC34-like PRRSV-2 whole-genome multi-target multiplex detection method V2 kit. Detailed Implementation

[0052] The embodiments of the present invention will be described in detail below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Modifications or substitutions to the methods, steps, or conditions of the present invention without altering its essence or departing from its spirit shall still fall within the scope of protection of the present invention.

[0053] The conventional experimental methods used in the following examples are based on Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition (Beijing: Science Press, 2002). The use of instruments should refer to their instruction manuals. Unless otherwise specified, all experimental materials, reagents, and consumables used in the examples of this invention are commercially available. The specific pathogenic biological materials used in this invention were actually sourced from the following sources: NADC30-like PRRSV-2SD17-36 (GenBank accession number: MH121061), JXA1-like PRRSV-2XJ17-5 (GenBank accession number: MK759853), NADC34-like PRRSV-2SDLY23-1742 (GenBank accession number: PQ373813) / BJ23-2652 (GenBank accession number: PX148100), VR2332-like PRRSV-2JSYC-2005-2 (GenBank accession number: MT746146), and PRRSV-1AHEU2024-2671 (GenBank accession number: PQ640355), all of which were isolated and preserved in our laboratory. The NADC34-like PRRSV-2rBJ1805-2 non-recombinant strain, with accession number CCTCC NO:V202250, has been published in Chinese patent application number 202210804949.1.

[0054] In this embodiment of the invention, other reagents used were as follows: RNase-free H2O was purchased from Solarbio; 2×Premix Ex Taq was purchased from TAKARA; QIAGEN RNAeasy Mini Kit was purchased from Qiagen Biotechnology Co., Ltd.; HiScript III 1st Strand cDNA Synthesis Kit was purchased from Novizumi Biotechnology Co., Ltd.; and DNA Marker was purchased from Zhejiang Boerjin Technology Co., Ltd. Primers used in the experiments were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. and General Biotech (Anhui) Co., Ltd., and probes were synthesized by Kunshan Pronopo Biotechnology Co., Ltd. and General Biotech (Anhui) Co., Ltd.

[0055] Example 1: Establishment of a genome-wide multi-target detection and recombination analysis method for NADC34-like PRRSV-2

[0056] 1. Design and screening of primers and probes for whole-genome detection of NADC34-like PRRSV-2

[0057] One hundred complete genome sequences covering all major lineages of PRRSV-1 and PRRSV-2 (including NADC30, NADC34, VR-2332, Lelystad, QYYZ, CH-1a, JXA1, etc.) were downloaded from the GenBank database. Multiple sequence alignment analysis was performed using the DNAMAN software. Based on highly conserved and specific gene regions in PRRSV strains, primers and probes (such as NADC34-like PRRSV-2 whole-genome detection primers) were designed. Figure 1 The regions selected from the whole genome include 80bp–300bp (number 1), 1000bp–1300bp (number 2), 1500bp–2200bp (number 3–6), 3800bp–7000bp (number 7–14), 7800bp–9100bp (number 15–16), 10000bp–10500bp (number 17–18), 11100bp–13500bp (number 19–24), and 13800bp–15000bp (number 25–28).

[0058] Primers were designed based on the relatively conserved regions described above, and the specific 28 primer-probe sequences are shown in Table 1. RNA extracted from NADC34-Like PRRSV2 (rBJ805-2 strain) using the QIAGEN RNAeasy Mini Kit was used as a template and reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit. Then, qPCR amplification and signal acquisition were performed using the reaction system shown in Table 2 and the reaction conditions shown in Table 3. Eight primer-probe combinations that did not amplify were removed, and 20 effective combinations were retained for subsequent experiments. The specific primer-probe sequences are shown in Table 1 (the 20 effective combinations include numbers 1-5, 7-8, 10, 12-15, 17-21, 24, 26, and 28).

[0059] Table 1. Primer and probe list for NADC34-like PRRSV-2 multiplex detection

[0060]

[0061]

[0062]

[0063]

[0064] Table 2 NADC34-Like PRRSV-2 Multiplex Detection Reaction System

[0065]

[0066] Table 3. NADC34-Like PRRSV-2 Multiplex Detection Amplification Program

[0067]

[0068] 2. Specificity verification

[0069] Using RNA extracted from different PRRSV strains (NADC34-like PRRSV-2rBJ1805-2, NADC30-like PRRSV-2SD17-36, HP-PRRSV2 XJ17-5, VR2332-like PRRSV-2JSYC-2005-2, and PRRSV-1AHEU2024-2671) extracted with the QIAGEN RNAeasy Mini Kit as templates, viral cDNA was prepared using the HiScript III 1stStrand cDNA Synthesis Kit to evaluate the specificity of this method. In this invention, real-time fluorescence PCR was used to detect five different PRRSV strains to evaluate the specificity of primer-probe combinations. Specific amplification conditions and systems are described in step 1. The results showed that some primer-probe combinations (numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, and 26 in Table 1) successfully detected specific fluorescent signals, such as... Figure 2A As shown, taking the N34-10260 group as an example, the results show that this primer-probe combination specifically amplifies only the rBJ1805-2 strain, indicating that they can accurately identify and amplify the nucleic acid sequence of the target strain. However, other primer-probe combinations exhibited non-specific amplification, such as... Figure 2B As shown, taking group N34-12110 as an example, this group amplified strains rBJ1805-2, SD17-36, and XJ17-5, meaning that additional non-specific fluorescent signals were generated during the detection process, which may interfere with the accuracy and reliability of the detection results. To ensure the rigor and effectiveness of subsequent experiments, it was decided to discard all primer-probe combinations that exhibited non-specific amplification, retaining only 18 primer-probe combinations that could generate specific fluorescent signals for subsequent experimental research (numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, and 26). This screening process is crucial for improving the specificity and accuracy of the detection method, laying a solid foundation for the accurate identification and analysis of PRRSV strains.

[0070] 3. Detection of recombinant strains

[0071] To verify the performance of the final selected primer-probe combination in detecting PRRSV recombinant strains, NADC34-like recombinant strain (SDLY23-1742) and NADC34-like non-recombinant strain (rBJ1805-2) were used as test samples. Real-time fluorescence PCR was used to detect these two strains; specific amplification conditions and systems are described in step 1. The method was found to accurately and efficiently distinguish between the recombinant and non-recombinant strains. Specifically, the quantitative detection results clearly showed the differences between the two strains. When detecting the NADC34-like non-recombinant strain (rBJ1805-2), the quantitative fluorescence curve and statistical analysis clearly presented the detection results. The results are as follows: Figure 3A As shown, the 18 primer-probe combinations numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, and 26 can achieve comprehensive and accurate detection of all target sites in the non-recombinant strain. Each target site can stably generate a recognizable fluorescent signal, indicating that these 18 primer-probe combinations have high sensitivity and specificity in the detection of non-recombinant strains, completely covering all predetermined detection targets and providing reliable basic data for subsequent analysis and research. However, when the detection target is changed to the NADC34-like recombinant strain (SDLY23-1742), the detection results show significant differences. For recombinant strains, as... Figure 3B As shown, no fluorescent signal was detected in the recombination region. This is because the changes in gene sequence during recombination prevent the fluorescent marker from binding or the fluorescent signal from being generated normally. Specific fluorescent signals were still detected in the non-recombined regions. Furthermore, we investigated the recombination status of the strain using other techniques, and the results showed that the recombination regions completely matched the regions lacking signals in the fluorescent detection, while the non-recombined parts also corresponded to the regions where fluorescent signals were detected. Figure 3C This suggests that the primer-probe combinations described above can not only indicate the occurrence of recombination events, but also accurately locate the distribution of NADC34-like fragments in recombinant strains. This result demonstrates that the primer-probe combinations used (numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, 26) have high specificity and accuracy in detecting PRRSV recombinant strains, providing strong technical support for PRRSV genetic variation research, epidemiological surveillance, and the formulation of prevention and control strategies.

[0072] 4. Testing and optimization of primer-probe combinations

[0073] In the design of multiplex detection primer-probe combinations, we fully considered and mitigated several key factors that could affect detection specificity. These factors include primer dimer formation, cross-reactions between different primers and probes, and differences in annealing temperatures between primers and probes. Through careful design and optimization, we strive to ensure that each primer-probe combination can efficiently and specifically amplify the target sequence in a multiplex detection environment.

[0074] During the experiment, we paired and grouped primer-probe combinations numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, and 26 for detection, resulting in a total of 153 combinations. For example, combination 1 included primer-probe combination number 1 and primer-probe combination number 5; combination 2 included primer-probe combination number 2 and primer-probe combination number 4; combination 3 included primer-probe combination number 8 and primer-probe combination number 10; combination 4 included primer-probe combination number 12 and primer-probe combination number 13; combination 5 included primer-probe combination number 14 and primer-probe combination number 18; combination 6 included primer-probe combination number 15 and primer-probe combination number 17; combination 7 included primer-probe combination number 19 and primer-probe combination number 24; combination 8 included primer-probe combination number 21 and primer-probe combination number 26, and so on.

[0075] However, despite the numerous measures taken during the design phase, interference between individual primer-probe combinations was still observed during the experiment. Figure 4A The example clearly illustrates this. Let's take one set as an example; specifically... Figure 4A The single detection results for a and b in the data show that when detected individually, both sets of primer probes (number 24: N34-13300 and number 26: N34-14390) stably produce fluorescent signals for all strains, indicating that the single detection system is effective in design and can accurately identify the target strains. However, when these two sets of primer probes are labeled in different channels and mixed together for dual detection, the results of combination 8' are as follows: Figure 4AAs shown in Figure c, the N34-14390P-HEX primer-probe combination (number 26) exhibited severe interference when detecting the rBJ1805-2 strain, resulting in no amplification signal. This interference may stem from competitive binding between primers and probes, steric hindrance, or insufficient compatibility of reaction conditions with different probes. This interference can lead to non-specific amplification or reduced amplification efficiency, thus affecting the accuracy and reliability of the detection results. To thoroughly resolve this issue, we conducted a detailed analysis and adjustment of these combinations. By adjusting the dual-combination method (e.g., discarding primer-probe combinations 24 (N34-13300) and 26 (N34-14390) of combination 8' and selecting primer-probe combinations 21 (N34-12550) and 26 (N34-14390) of combination 8; correspondingly discarding primer-probe combinations 19 (N34-11360) and 21 (N34-12550) of combination 7' ​​and selecting combination 7 including primer-probe combinations 19 (N34-11360) and 24 (N34-13300) of combination 7') and discarding primer-probe combinations that cannot solve the interference problem (numbers 3: N34-1630 and 7: 3930), from Figure 4B As you can see, we have successfully solved the problem of mutual interference between these combinations.

[0076] After a series of optimizations and adjustments, the final 16 primer-probe combinations (numbered 1, 2, 4, 5, 8, 10, 12-15, 17-19, 21, 24, and 26) demonstrated excellent specificity and sensitivity in multiplex detection, accurately amplifying the target sequence while avoiding non-specific amplification. This improvement not only enhanced the overall performance of the detection method but also laid a solid foundation for subsequent experimental research and practical applications.

[0077] 5. Development and iteration of reagent kits

[0078] Based on the above optimization scheme, we have developed a series of kits, the main components of which include negative controls, positive controls, nuclease-free water, RT-PCR reaction solution, reverse transcriptase and Taq enzyme mixture, and single or double mixtures of 16 primer probes. For example, the developed V1 kit pairs 16 primer probes to form 8 double detection combinations (see details for specific combination methods). Figure 5 After assembly, it underwent comprehensive testing.

[0079] like Figure 5 As shown, the quantitative amplification curves and statistical analysis results clearly demonstrate the detection performance of the V1 kit. From Figure 5As can be seen, all combinations of the V1 kit stably generated specific fluorescence signals, and the intensity and pattern of these signals were highly consistent with the intended design target. This indicates that the V1 kit exhibited excellent specificity and sensitivity during the detection process, accurately and efficiently completing the target detection task while avoiding interference from non-specific amplification. To further verify whether the different primer and probe synthesis companies would affect the detection performance of the kit, we changed to another synthesis company (General Biotechnology (Anhui) Co., Ltd.), resynthesized the primers and probes, and prepared the V2 kit. After rigorous testing, we found that the detection results of the V2 kit (e.g., ...) were significantly better than those of the V1 kit. Figure 6 As shown in the image, the kit is completely identical to the V1 kit, indicating that the source of primer and probe synthesis did not significantly affect the kit's performance. This result further confirms the stability and reliability of the optimized scheme, providing a strong basis for the finalization of the kit and subsequent large-scale production. Ultimately, we decided to use this optimized scheme to prepare this kit to ensure its accuracy and reliability in practical applications.

Claims

1. A primer-probe combination for NADC34-like PRRSV-2, characterized in that, The primer-probe combination is as follows: For example, the upstream primer N34-130F shown in SEQ ID NO.1, the probe N34-130P shown in SEQ ID NO.2, and the downstream primer N34-130R shown in SEQ ID NO.3; For example, the upstream primer N34-1970F shown in SEQ ID NO.10, the probe N34-1970P shown in SEQ ID NO.11, and the downstream primer N34-1970R shown in SEQ ID NO.12; For example, the upstream primer N34-4760F shown in SEQ ID NO.13, the probe N34-4760P shown in SEQ ID NO.14, and the downstream primer N34-4760R shown in SEQ ID NO.15; For example, the upstream primer N34-6610F shown in SEQ ID NO.22, the probe N34-6610P shown in SEQ ID NO.23, and the downstream primer N34-6610R shown in SEQ ID NO.24; For example, the upstream primer N34-8090F shown in SEQ ID NO.28, the probe N34-8090P shown in SEQ ID NO.29, and the downstream primer N34-8090R shown in SEQ ID NO.30; For example, the upstream primer N34-10260F shown in SEQ ID NO.31, the probe N34-10260P shown in SEQ ID NO.32, and the downstream primer N34-10260R shown in SEQ ID NO.33; For example, the upstream primer N34-12550F shown in SEQ ID NO.40, the probe N34-12550P shown in SEQ ID NO.41, and the downstream primer N34-12550R shown in SEQ ID NO.42; For example, the upstream primer N34-13300F shown in SEQ ID NO.43, the probe N34-13300P shown in SEQ ID NO.44, and the downstream primer N34-13300R shown in SEQ ID NO.45; For example, the upstream primer N34-1160F shown in SEQ ID NO.4, the probe N34-1160P shown in SEQ ID NO.5, and the downstream primer N34-1160R shown in SEQ ID NO.6; For example, the upstream primer N34-1750F shown in SEQ ID NO.7, the probe N34-1750P shown in SEQ ID NO.8, and the downstream primer N34-1750R shown in SEQ ID NO.9; For example, the upstream primer N34-5460F shown in SEQ ID NO.16, the probe N34-5460P shown in SEQ ID NO.17, and the downstream primer N34-5460R shown in SEQ ID NO.18; For example, the upstream primer N34-6080F shown in SEQ ID NO.19, the probe N34-6080P shown in SEQ ID NO.20, and the downstream primer N34-6080R shown in SEQ ID NO.21; For example, the upstream primer N34-6700F shown in SEQ ID NO.25, the probe N34-6700P shown in SEQ ID NO.26, and the downstream primer N34-6700R shown in SEQ ID NO.27; For example, the upstream primer N34-10270F shown in SEQ ID NO.34, the probe N34-10270P shown in SEQ ID NO.35, and the downstream primer N34-10270R shown in SEQ ID NO.36; For example, the upstream primer N34-11360F shown in SEQ ID NO.37, the probe N34-11360P shown in SEQ ID NO.38, and the downstream primer N34-11360R shown in SEQ ID NO.39; For example, the upstream primer N34-14390F shown in SEQ ID NO.46, the probe N34-14390P shown in SEQ ID NO.47, and the downstream primer N34-14390R shown in SEQ ID NO.

48.

2. The primer-probe combination according to claim 1, characterized in that, The concentrations of the primers and probes were both 5–20 μM.

3. The use of the primer-probe combination according to any one of claims 1 to 2 in the preparation of a detection kit capable of simultaneously detecting the genome of NADC34-like PRRSV-2 strains.

4. A detection kit capable of simultaneously detecting the genome of NADC34-like PRRSV-2 strains, characterized in that, The detection kit includes the primer-probe combination as described in any one of claims 1 to 2.

5. The detection kit according to claim 4, characterized in that, The test kit also includes other test reagents required for real-time quantitative PCR.

6. The detection kit according to claim 4, characterized in that, The test kit includes 2×Premix ExTaq and RNase-Free H2O.

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