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

Abstract

The invention discloses a primer and probe combination for NADC34-like PRRSV-2 whole genome multi-target detection and a detection kit for NADC34-like PRRSV-2 whole genome multi-target detection. According to the whole genome multi-target detection, multiple targets can be detected at the same time, the detection efficiency is remarkably improved, the single detection cost is reduced, and whether the virus is recombined or not is identified. The technology has obvious advantages in market competition due to high specificity and high sensitivity. Through multiple detections covering the whole genome, the technology can complete detection of a plurality of gene targets in one reaction, so that the repeated use of reagents and equipment is reduced, and the detection cost is remarkably reduced. Therefore, the accurate and rapid detection method is more economical and feasible, and is beneficial to wide popularization and application in the pig industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of viral nucleic acid detection, and particularly relates to a primer probe combination and a detection kit for NADC34-like PRRSV-2 whole genome multi-target detection. BACKGROUND

[0002] In recent years, NADC34-like porcine reproductive and respiratory syndrome virus (NADC34-like PRRSV-2) has become the main epidemic strain in the pig industry. Its genome frequently undergoes recombination events, resulting in significant differences in the pathogenicity of the strain. Existing research shows that NADC34-like strains often undergo complex recombination with NADC30-like, JXA1-like and other native strains to form chimeric viruses. These recombination events significantly increase the risk of virus transmission and exacerbate the difficulty of prevention and control. Therefore, developing a detection technology that can quickly identify NADC34-like recombinant strains is crucial for epidemic monitoring. Most current RT-qPCR methods (such as TaqMan probe method based on ORF7 or Nsp2 gene) are mainly used for PRRSV general detection or typing. Although these methods have high sensitivity and good specificity, their design targets are mostly conserved regions, which cannot effectively identify the unique recombination characteristics of NADC34-like strains. For example, some methods only use ORF5 RFLP typing (1-7-4 mode) to determine NADC34-like strains, but genome phylogenetic analysis shows that such strains may carry recombination fragments from other lineages, resulting in a mismatch between the typing results and the true recombination status. Existing recombination detection mainly relies on next-generation sequencing (NGS) and bioinformatics analysis (such as RDP software). Although these methods can comprehensively analyze recombination sites, they require several days and are costly, making them difficult to be used for large-scale rapid screening in clinical settings. Recently, a SYBR Green RT-qPCR method targeting NADC34-like strains has been developed, but due to the highly variable nature of NADC34-like strains, this method may miss detection due to variation in the single target point, and cannot distinguish between recombinant and non-recombinant strains.

[0003] The first inventor of the present application, Chen Nanhua, has been committed to the research of PRRSV differential diagnosis method since 2006. A number of SCI papers related to PRRSV detection have been published (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 simultaneously detection and differentiation of porcine reproductive and respiratory syndrome viruses. Transboundary and Emerging Diseases, 66(6):2271-2278. DOI: 10.1111 / tbed.13276.), and a number of invention patents related to PRRSV fluorescence quantitative PCR differential detection have been applied for (PRRSV classical strain and highly pathogenic variant strain duplex real-time fluorescence RT-PCR differential 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 accumulated in the field of primer probe design, when designing primer probe combinations, most potential factors that may interfere with multiplex differential detection have been fully considered and avoided as much as possible. These factors include but are not limited to the dimers that may be formed between primer probe sequences, the mutual interference between primer probe combinations of different targets, and the difference in annealing temperature, etc. However, although careful consideration and optimization have been made in the design stage, many challenges have still been encountered in the actual test process, such as the mutual inhibition between primer probes, the synergistic effect between primer probes, and more complexly, some primer probe combinations are incompatible with any other primer probe combinations, etc. leading to no amplification signal, etc. This phenomenon may be related to the serious mutual interference between primer probes.This interference can be caused by high homology, complementarity or steric hindrance between primer probe sequences, resulting in the failure of primer probe to normally bind to the target template during the reaction, so that the amplification reaction cannot be started. SUMMARY

[0004] The application aims to solve the technical problems of the prior art, such as the single or few target points being easy to mutate, the missed detection, and the inability to identify whether the strain has recombination, etc. The application successfully develops a multiplex real-time fluorescent quantitative PCR technology based on a whole genome multi-target detection strategy through innovative experimental design and system optimization, and provides a plurality of primer probe combinations for accurately detecting the genomic characteristics of NADC34-like PRRSV-2 strain, so as to significantly improve the recognition ability of the recombinant strain by synchronously detecting a plurality of conserved regions of the whole genome of the virus and combining the optimized primer probe combination design.

[0005] The application also aims to solve the technical problems of providing a high-sensitivity and high-specificity detection kit containing the primer probe combination, and providing important technical support for detecting the recombination evolution dynamics of NADC34-like strain and formulating a precise prevention and control strategy.

[0006] Technical scheme: In order to solve the above technical problems, the application provides a primer probe combination covering the whole genome of NADC34-like PRRSV-2, which includes any one or several of the following combinations:

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

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

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

[0010] an upstream primer N34-1970F as set forth in SEQ ID NO. 10, a probe N34-1970P as set forth in SEQ ID NO. 11, and a downstream primer N34-1970R as set forth in SEQ ID NO. 12; this primer probe combination is designated as No. 5;

[0011] an upstream primer N34-4760F as set forth in SEQ ID NO. 13, a probe N34-4760P as set forth in SEQ ID NO. 14, and a downstream primer N34-4760R as set forth in SEQ ID NO. 15; this primer probe combination is designated as No. 8;

[0012] an upstream primer N34-5460F as set forth in SEQ ID NO. 16, a probe N34-5460P as set forth in SEQ ID NO. 17, and a downstream primer N34-5460R as set forth in SEQ ID NO. 18; this primer probe combination is designated as No. 10;

[0013] an upstream primer N34-6080F as set forth in SEQ ID NO. 19, a probe N34-6080P as set forth in SEQ ID NO. 20, and a downstream primer N34-6080R as set forth in SEQ ID NO. 21; this primer probe combination is designated as No. 12;

[0014] an upstream primer N34-6610F as set forth in SEQ ID NO. 22, a probe N34-6610P as set forth in SEQ ID NO. 23, and a downstream primer N34-6610R as set forth in SEQ ID NO. 24; this primer probe combination is designated as No. 13;

[0015] an upstream primer N34-6700F as set forth in SEQ ID NO. 25, a probe N34-6700P as set forth in SEQ ID NO. 26, and a downstream primer N34-6700R as set forth in SEQ ID NO. 27; this primer probe combination is designated as No. 14;

[0016] an upstream primer N34-8090F as set forth in SEQ ID NO. 28, a probe N34-8090P as set forth in SEQ ID NO. 29, and a downstream primer N34-8090R as set forth in SEQ ID NO. 30; this primer probe combination is designated as No. 15;

[0017] an upstream primer N34-10260F as set forth in SEQ ID NO. 31, a probe N34-10260P as set forth in SEQ ID NO. 32, and a downstream primer N34-10260R as set forth in SEQ ID NO. 33; this primer-probe combination is designated as No. 17;

[0018] an upstream primer N34-10270F as set forth in SEQ ID NO. 34, a probe N34-10270P as set forth in SEQ ID NO. 35, and a downstream primer N34-10270R as set forth in SEQ ID NO. 36; this primer-probe combination is designated as No. 18;

[0019] an upstream primer N34-11360F as set forth in SEQ ID NO. 37, a probe N34-11360P as set forth in SEQ ID NO. 38, and a downstream primer N34-11360R as set forth in SEQ ID NO. 39; this primer-probe combination is designated as No. 19;

[0020] an upstream primer N34-12550F as set forth in SEQ ID NO. 40, a probe N34-12550P as set forth in SEQ ID NO. 41, and a downstream primer N34-12550R as set forth in SEQ ID NO. 42; this primer-probe combination is designated as No. 21;

[0021] an upstream primer N34-13300F as set forth in SEQ ID NO. 43, a probe N34-13300P as set forth in SEQ ID NO. 44, and a downstream primer N34-13300R as set forth in SEQ ID NO. 45; this primer-probe combination is designated as No. 24;

[0022] an upstream primer N34-14390F as set forth in SEQ ID NO. 46, a probe N34-14390P as set forth in SEQ ID NO. 47, and a downstream primer N34-14390R as set forth in SEQ ID NO. 48; this primer-probe combination is designated as No. 26.

[0023] As preferred, the present application further comprises any combination of the 16 groups of primer-probe combinations designated as No. 1, 2, 4, 5, 8, 10, 12-15, 17-19, 21, 24, 26.

[0024] When performing singleplex detection, any one of the above groups of primer-probe combinations can be arbitrarily selected;

[0025] When performing duplex detection, the present application also includes any two of the 16 groups of primer probe combinations numbered 1, 2, 4, 5, 8, 10, 12-15, 17-19, 21, 24, 26 in the form of duplex detection. Among them, there are 120 combinations of any two combinations.

[0026] As a preferred, the two two combinations of the duplex detection of the present application include: combination 1: primer probe combination numbered 1 (N34-130) and numbered 5 (N34-1970); combination 2: primer probe combination numbered 2 (N34-1160) and numbered 4 (N34-1750); combination 3: primer probe combination numbered 8 (N34-4760) and numbered 10 (N34-5460); combination 4: primer probe combination numbered 12 (N34-6080) and numbered 13 (N34-6610); combination 5: primer probe combination numbered 14 (N34-6700) and numbered 18 (N34-10270); combination 6: primer probe combination numbered 15 (N34-8090) and numbered 17 (N34-10260); combination 7: primer probe combination numbered 19 (N34-11360) and numbered 24 (N34-13300); combination 8: primer probe combination numbered 21 (N34-12550) and numbered 26 (N34-14390).

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

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

[0029] Among them, the concentration of the primer and the probe is 5-20 μM. As a preferred, the concentration of the primer and the probe is 10 μM.

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

[0031] The present application also includes a multiplex detection kit for simultaneously detecting the whole genome of the NADC34-like PRRSV-2 strain, which comprises the primer probe combination.

[0032] The multiplex detection kit further comprises other detection reagents required for multiplex PCR.

[0033] The multiplex PCR comprises real-time fluorescent quantitative PCR.

[0034] The multiplex detection kit comprises 2xPremix Ex Taq and RNase Free H2O.

[0035] The amplification procedure of the multiplex detection kit is as follows: 95°C for 30s for 1 cycle; 95°C for 5s, 60°C for 1min for 40 cycles.

[0036] Advantages: Compared with the prior art, the present application has the following advantages: The present application can effectively distinguish NADC34-like PRRSV-2 from other PRRSV strains through whole genome multi-target detection, significantly improves the accuracy of detection, and reduces misdiagnosis and missed diagnosis caused by virus variation. The present application realizes multiplex detection through primer probe combination, can detect multiple gene targets at one time, greatly improves the detection efficiency, and reduces the detection cost, so that large-scale detection is more economical and feasible. Since multiple key gene regions of the whole genome of the virus are covered, the recombination of each gene of NADC34-like PRRSV-2 can be effectively detected, which provides important data support for the evolution research and prevention and control of the virus. In summary, the present application overcomes the shortcomings of the prior art through whole genome multi-target detection and recombination analysis technology, provides stronger technical support for the detection, genetic evolution analysis and effective prevention and control of NADC34-like PRRSV-2, and has important practical significance and application value. Specifically, the present application includes the following aspects:

[0037] 1. The specific primer pairs and probes designed for multiple key gene regions of the whole genome of NADC34-like PRRSV-2 can effectively distinguish NADC34-like PRRSV-2 from other lineage strains. The traditional single-target detection method is prone to false negatives when facing high-frequency variation and recombination of viruses, while the multi-target strategy covers the conserved region and the variable region, significantly improves the detection accuracy, and reduces the breeding loss caused by missed detection and misjudgment.

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

[0039] 3、Multiple detection reduces reagent consumption and equipment reuse, reduces the cost of single sample to 1 / 2 of the conventional method. Compared with traditional RT-PCR or ordinary RT-qPCR, the method uses a premixed reaction system or TaqMan probe design, simplifies the operation process and shortens the detection time, while avoiding the problem of non-specific amplification of SYBR Green method, and is more suitable for large-scale clinical rapid detection and precise analysis requirements.

[0040] 4、At present, NADC34-like strains are prevalent in many places, but the protective effect of existing commercial vaccines is limited. The present application can provide key technical support for early monitoring, vaccine development evaluation and precise prevention and control of the strain, and reduce economic losses caused by false detection or missed detection.

[0041] 5、The present application realizes the simultaneous detection of multiple targets of NADC34-like PRRSV-2. The application of this technology has important monitoring value for pig farms with PRRSV infection. Through regular detection, the genetic characteristic changes of the virus in the pig population can be monitored in real time. If the detected combination changes in a certain detection, it usually indicates that a new strain may have been introduced into the pig population. The timely discovery of such changes can provide key data for the pig farm to take preventive measures as soon as possible to prevent the new strain from spreading widely in the pig population, thereby effectively reducing the economic losses caused by the epidemic. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is an example of a specific conserved region screening design of the whole genome of NADC30-like PRRSV-2;

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

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

[0045] Figure 3A is a test result graph of the detection performance of the whole genome multi-target detection method of NADC34-like PRRSV2 on non-recombinant strains (rBJ1805-2);

[0046] Figure 3B is a test result graph of the detection performance of the whole genome multi-target detection method of NADC34-like PRRSV2 on recombinant strains (SDLY23-1742);

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

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

[0049] Figure 4B is a graph of the results of a combination optimization example of a whole genome multi-target multiplex detection method for NADC34-like PRRSV-2;

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

[0051] Figure 6 is a graph of the results of a V2 kit test of a whole genome multi-target multiplex detection method for NADC34-like PRRSV-2. DETAILED DESCRIPTION

[0052] Embodiments of the present application will be described in detail below with reference to examples. The following examples are intended to be illustrative only and are not intended to limit the scope of the present application. Modifications or substitutions to the methods, steps or conditions of the present application can be made without changing the nature of the present application or departing from the spirit of the present application, and still fall within the scope of the present application.

[0053] The general experimental methods in the following examples refer to Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd edition (Beijing: Science Press, 2002), and the use of instruments refers to the instrument operation manual. Unless otherwise specified, the experimental materials, reagents, consumables, etc. used in the examples of the present application can be commercially available. The specific pathogenic biological materials used in the present application are actually derived as follows: NADC30-like PRRSV-2 SD17-36 (GenBank accession number: MH121061) strain, JXA1-like PRRSV-2 XJ17-5 (GenBank accession number: MK759853) strain, NADC34-like PRRSV-2 SDLY23-1742 (GenBank accession number: PQ373813) strain / BJ23-2652 (GenBank accession number: PX148100) strain, VR2332-like PRRSV-2 JSYC-2005-2 (GenBank accession number: MT746146) strain, and PRRSV-1 AHEU2024-2671 (GenBank accession number: PQ640355) strain are isolated and preserved by the laboratory. The NADC34-like PRRSV-2 rBJ1805-2 non-recombinant strain has the preservation number CCTCC NO: V202250 and has been disclosed in the Chinese patent application with the application number 202210804949.1.

[0054] In the examples of the present application, other reagents used include RNase Free H2O purchased from Solarbio Company; 2xPremix Ex Taq purchased from TAKARA Company; QIAGEN RNAeasy Mini Kit purchased from KANGJIE Biological Engineering Co., Ltd.; HiScript III 1st Strand cDNA Synthesis Kit purchased from Novozyme Biological Technology Co., Ltd.; and DNA Marker purchased from Zhejiang Boerjin Technology Co., Ltd. The primers used in the test were synthesized by Suzhou Jinweizhi Biological Technology Co., Ltd. and Universal Biological (Anhui) Co., Ltd., and the probes were synthesized by Kunshan Punopu Biological Technology Co., Ltd. and Universal Biological (Anhui) Co., Ltd.

[0055] Example 1 Establishment of whole genome multi-target detection and recombinant 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] The complete genome sequences of 100 PRRSV-1 and PRRSV-2 strains covering all major lineages (including NADC30, NADC34, VR-2332, Lelystad, QYYZ, CH-1a, JXA1, etc.) were downloaded from the GenBank database, and multiple sequence alignment analysis was performed using sequence alignment software DNAMAN. Based on the highly conserved and specific gene regions in PRRSV strains, primers and probes for NADC34-like PRRSV-2 whole genome detection were designed (as shown in Figure 1 ) The selected regions on the whole genome include 80bp-300bp (No. 1), 1000bp-1300bp (No. 2), 1500bp-2200bp (No. 3- No. 6), 3800bp-7000bp (No. 7- No. 14), 7800bp-9100bp (No. 15- No. 16), 10000bp-10500bp (No. 17- No. 18), 11100bp-13500bp (No. 19- No. 24), 13800bp-15000bp (No. 25- No. 28).

[0058] According to the above relatively conserved regions, 28 groups of primers and probes were designed. The specific sequences of the primers and probes are shown in Table 1. The RNA of NADC34-Like PRRSV2 (rBJ805-2 strain) extracted by QIAGEN RNAeasy Mini Kit was used as the template, and the HiScript III 1st Strand cDNA Synthesis Kit was used for reverse transcription to cDNA. Then, qPCR amplification and signal collection were performed by the reaction system shown in Table 2 and the reaction conditions shown in Table 3. Eight groups of primer-probe combinations without amplification signal were removed, and 20 groups of effective combinations were retained for subsequent experiments. The specific primer and probe sequences are shown in Table 1 (20 groups of effective combinations include No. 1-5, 7-8, 10, 12-15, 17-21, 24, 26, 28).

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

[0060]

[0061]

[0062]

[0063]

[0064] Table 2. Reaction system for NADC34-Like PRRSV-2 multiplex detection

[0065]

[0066] Table 3 NADC34-Like PRRSV-2 multiplex detection amplification procedure

[0067]

[0068] 2. Specificity verification

[0069] The RNA of different PRRSV strains (NADC34-like PRRSV-2 rBJ1805-2 strain, NADC30-like PRRSV-2 SD17-36 strain, HP-PRRSV2 XJ17-5 strain, VR2332-like PRRSV-2 JSYC-2005-2 strain and PRRSV-1 AHEU2024-2671 strain) extracted by QIAGEN RNAeasy Mini Kit was used as a template to prepare viral cDNA using HiScript III 1stStrand cDNA Synthesis Kit to evaluate the specificity of the method. In this application, real-time fluorescent PCR method was used to detect 5 different PRRSV strains to evaluate the specificity of the primer probe combination. The specific amplification conditions and amplification system are shown in step 1. The results show that some primer probe combinations (1-5, 7-8, 10, 12-15, 17-19, 21, 24, 26 in Table 1) can successfully detect specific fluorescent signals, such as Figure 2A As shown in N34-10260 group, the results show that this primer probe combination only specifically amplifies the rBJ1805-2 strain, indicating that they can accurately recognize and amplify the nucleic acid sequence of the target strain. However, some other primer probe combinations have non-specific amplification phenomenon, such as Figure 2B As shown in N34-12110 group, this group amplifies rBJ1805-2 strain, SD17-36 strain and XJ17-5 strain, that is, additional non-specific fluorescent signals are generated during the detection process, which may interfere with the accuracy and reliability of the detection results. In order to ensure the rigor and effectiveness of subsequent experiments, all primer probe combinations that appear non-specific amplification are discarded, and only 18 groups of primer probe that can produce specific fluorescent signals (1-5, 7-8, 10, 12-15, 17-19, 21, 24, 26) are reserved for subsequent experimental research. This screening process is crucial to improve the specificity and accuracy of the detection method, and lays a solid foundation for the accurate identification and analysis of PRRSV strains in the follow-up.

[0070] 3. Detection of recombinant strains

[0071] To verify the performance of the final screened 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. The two strains were detected by real-time fluorescent PCR method, and the specific amplification conditions and amplification system are shown in step 1. It is found that this method can accurately and efficiently distinguish recombinant strains from non-recombinant strains. Specifically, the quantitative detection results clearly show the difference between the two strains, and the quantitative fluorescent curve and statistical analysis chart clearly show the detection results when detecting NADC34-like non-recombinant strain (rBJ1805-2). The results are shown in Figure 3A As shown in the table, the 18 groups of 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 points of non-recombinant strains, and each target point can stably produce identifiable fluorescent signals, which indicates that the above 18 groups of primer probe combinations have high sensitivity and specificity in detecting non-recombinant strains, and can completely cover all predetermined detection targets, providing reliable basic data for subsequent analysis and research. However, when the detection object changes to NADC34-like recombinant strain (SDLY23-1742), the detection results show significant differences. As shown in Figure 3B For recombinant strains, as shown in the table, no fluorescent signal was detected in the recombinant segment. This is because the change of gene sequence during recombination leads to the failure of fluorescent label binding or the failure of fluorescent signal production. In the non-recombinant region, specific fluorescent signals can still be detected. In addition, we further explored the recombination of the strain by other technical means, and the results showed that the recombinant region was completely consistent with the region where the signal was missing in the fluorescent detection, and the part that did not recombine also corresponded to the region where the fluorescent signal was detected Figure 3C This suggests that the above primer probe combinations of the present application not only indicate the occurrence of recombination events, but also accurately locate the distribution of NADC34-like fragments in recombinant strains. This result shows that the primer probe combinations (numbered 1-5, 7-8, 10, 12-15, 17-19, 21, 24, and 26) used have high specificity and accuracy in detecting PRRSV recombinant strains, and can provide strong technical support for the genetic variation research, epidemiological monitoring, and prevention and control strategy of PRRSV.

[0072] 4. Test and optimization of primer probe combination

[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 can stably produce specific fluorescent signals, and the intensity and pattern of these signals are highly consistent with the expected design goals. This indicates that the V1 kit exhibits excellent specificity and sensitivity during detection, can accurately and efficiently complete the target detection task, and at the same time avoids the interference of non-specific amplification. In order to further verify whether the different synthesis companies of the primers and probes will affect the detection performance of the kit, we replaced another synthesis company (General Biological (Anhui) Co., Ltd.) to re-synthesize the primers and probes, and prepared the V2 kit. After strict testing, we found that the detection results (such as Figure 6 shown) of the V2 kit were completely consistent with those of the V1 kit, indicating that the synthesis source of the primers and probes did not significantly affect the performance of the kit. This result further confirms the stability and reliability of the optimization scheme, providing a strong basis for the final standardization and subsequent large-scale production of the kit. Finally, we determined to use the optimization 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 whole genome coverage, characterized in that, The primer probe combination comprises any one or several of the following combinations: an upstream primer N34-130F as shown in SEQ ID NO. 1, a probe N34-130P as shown in SEQ ID NO. 2, and a downstream primer N34-130R as shown in SEQ ID NO. 3; an upstream primer N34-1160F as shown in SEQ ID NO. 4, a probe N34-1160P as shown in SEQ ID NO. 5, and a downstream primer N34-1160R as shown in SEQ ID NO. 6; an upstream primer N34-1750F as shown in SEQ ID NO. 7, a probe N34-1750P as shown in SEQ ID NO. 8, and a downstream primer N34-1750R as shown in SEQ ID NO. 9; an upstream primer N34-1970F as shown in SEQ ID NO. 10, a probe N34-1970P as shown in SEQ ID NO. 11, and a downstream primer N34-1970R as shown in SEQ ID NO. 12; an upstream primer N34-4760F as shown in SEQ ID NO. 13, a probe N34-4760P as shown in SEQ ID NO. 14, and a downstream primer N34-4760R as shown in SEQ ID NO. 15; an upstream primer N34-5460F as shown in SEQ ID NO. 16, a probe N34-5460P as shown in SEQ ID NO. 17, and a downstream primer N34-5460R as shown in SEQ ID NO. 18; an upstream primer N34-6080F as shown in SEQ ID NO. 19, a probe N34-6080P as shown in SEQ ID NO. 20, and a downstream primer N34-6080R as shown in SEQ ID NO. 21; an upstream primer N34-6610F as shown in SEQ ID NO. 22, a probe N34-6610P as shown in SEQ ID NO. 23, and a downstream primer N34-6610R as shown in SEQ ID NO. 24; an upstream primer N34-6700F as shown in SEQ ID NO. 25, a probe N34-6700P as shown in SEQ ID NO. 26, and a downstream primer N34-6700R as shown in SEQ ID NO. 27; an upstream primer N34-8090F as shown in SEQ ID NO. 28, a probe N34-8090P as shown in SEQ ID NO. 29, and a downstream primer N34-8090R as shown in SEQ ID NO. 30; an upstream primer N34-10260F as shown in SEQ ID NO. 31, a probe N34-10260P as shown in SEQ ID NO. 32, and a downstream primer N34-10260R as shown in SEQ ID NO. 33; an upstream primer N34-10270F as set forth in SEQ ID NO. 34, a probe N34-10270P as set forth in SEQ ID NO. 35, and a downstream primer N34-10270R as set forth in SEQ ID NO. 36; an upstream primer N34-11360F as set forth in SEQ ID NO. 37, a probe N34-11360P as set forth in SEQ ID NO. 38, and a downstream primer N34-11360R as set forth in SEQ ID NO. 39; an upstream primer N34-12550F as set forth in SEQ ID NO. 40, a probe N34-12550P as set forth in SEQ ID NO. 41, and a downstream primer N34-12550R as set forth in SEQ ID NO. 42; an upstream primer N34-13300F as set forth in SEQ ID NO. 43, a probe N34-13300P as set forth in SEQ ID NO. 44, and a downstream primer N34-13300R as set forth in SEQ ID NO. 45; an upstream primer N34-14390F as set forth in SEQ ID NO. 46, a probe N34-14390P as set forth in SEQ ID NO. 47, and a downstream primer N34-14390R as set forth in SEQ ID NO.

48.

2. The primer probe combination of claim 1, wherein The probes are labeled with any one of fluorescein, and the other probe must be labeled with a different detection channel fluorescein selected from any one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 fluorescein.

3. The primer probe combination of claim 1, wherein one or more of the NADC34-like PRRSV-2-SDLY23-1742 strain, the NADC34-like PRRSV-2-BJ23-2652 strain, and the NADC34-like PRRSV-2 rBJ1805-2 strain.

4. The primer probe combination of claim 1, wherein The concentrations of the primers and the probes are both 5-20 μM.

5. Use of the primer-probe combination of any one of claims 1-4 in the preparation of a multiplex detection kit capable of simultaneously detecting the whole genome of the NADC30-like PRRSV-2 strain.

6. A multiplex detection kit capable of simultaneously detecting the whole genome of NADC34-like PRRSV-2 strain, characterized in that, The multiplex detection kit comprises the primer-probe combination of any one of claims 1-4.

7. The multiplex detection kit according to claim 6, wherein The multiplex detection kit further comprises other detection reagents required for multiplex PCR.

8. The multiplex detection kit according to claim 7, wherein The multiplex PCR comprises real-time fluorescent quantitative PCR.

9. The multiplex detection kit according to claim 7, wherein The multiplex detection kit comprises 2x Premix Ex Taq and RNase Free H2O.

10. The multiplex detection kit according to claim 6, wherein The amplification procedure of the multiplex detection kit is: 95℃ for 30 s for 1 cycle; 95℃ for 5 s, 60℃ for 1 min for 40 cycles.

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