Primer probe combination and multiple detection kit for NADC30-like PRRSV-2 whole genome coverage

Abstract

The invention discloses a primer probe combination and a multiple detection kit for covering a whole genome of NADC30-like PRRSV-2 (Nicotinamide adenine dinucleotide 30-like PRRSV-2). Compared with the prior art, the method has the advantages that a whole genome multi-target detection strategy is adopted, and the recombination condition of NADC30-like PRRSV-2 can be effectively detected and analyzed; multiple detection is achieved, multiple gene targets can be detected at the same time through single reaction, the detection efficiency is remarkably improved, and the detection cost of a single gene is reduced. By designing the primer probes covering the key area of the whole genome, the virus recombination characteristics can be accurately identified, and important data support is provided for virus evolution research and prevention and control strategy formulation. The technology is simple and convenient to operate, reliable in result and suitable for large-scale screening of clinical samples.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of viral nucleic acid detection, and particularly relates to a primer probe combination for full-genome coverage of NADC30-like PRRSV-2 and a multiplex detection kit. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes significant economic losses to the global pig industry. In recent years, the prevalence of NADC30-like PRRSV-2 has brought new challenges to the pig industry. NADC30-like PRRSV-2 has high genetic diversity and recombination ability, which makes the traditional detection method have many limitations in detecting and identifying the virus. Therefore, it is of great practical significance to develop a multi-target multiplex detection RT-qPCR technology that can cover the whole genome of NADC30-like PRRSV-2.

[0003] Single-target detection cannot effectively distinguish NADC30-like PRRSV-2 from other PRRSV strains. If the detection site mutates or recombines, it will lead to a high false negative rate of the detection result. Moreover, NADC30-like PRRSV-2 has high recombination ability, and single-target detection cannot detect the recombination of the virus.

[0004] Although the existing qPCR detection method can detect at most 5 target points at the same time, these target points are usually concentrated in limited gene regions and cannot comprehensively cover the whole genome of PRRSV. Moreover, the existing qPCR detection method cannot effectively detect the recombination in the viral genome, but the analysis of the recombination is particularly important for NADC30-like PRRSV-2.

[0005] 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. j viromet. 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 obtained a number of patents related to PRRSV fluorescent quantitative PCR differential detection (PRRSV classic strain and highly pathogenic variant strain double real-time fluorescent RT-PCR differential detection method, ZL200910077704.8; Porcine reproductive and respiratory syndrome virus universal type real-time fluorescent RT-PCR detection method and kit, ZL201110073381.2). With more than ten years of rich experience 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 dimers that may be formed between primer probe sequences, mutual interference between primer probe combinations of different targets, and differences 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 mutual inhibition between primer probes, synergy between primer probes, and more complexly, some primer probe combinations are not compatible with any other primer probe combinations, etc. leading to no amplification signal, etc. This phenomenon may be related to serious mutual interference between primer probes. This interference may be caused by factors such as high homology, complementarity or steric hindrance between primer probe sequences, leading to the failure of primer probes to normally bind to the target template during the reaction, and thus the failure to initiate the amplification reaction. SUMMARY

[0006] The application aims to provide a primer probe combination that can effectively detect NADC30-like PRRSV-2 for whole genome coverage.

[0007] The application further aims to provide an application of the primer probe combination in a multiplex detection kit.

[0008] The application finally aims to provide a multiplex detection kit containing the primer probe combination for holographic multi-target multiplex amplification analysis of NADC30-like PRRSV-2 epidemic strain, which can effectively identify the genetic recombination characteristics of NADC30-like PRRSV-2, and has high detection accuracy and high sensitivity.

[0009] Technical solution: In order to solve the above technical problems, the present application provides a NADC30-like PRRSV-2 full genome covering primer probe combination, the primer probe combination includes any one or several of the following combinations:

[0010] The upstream primer N30-1150F as shown in SEQ ID NO. 1, the probe N30-1150P as shown in SEQ ID NO. 2 and the downstream primer N30-1150R as shown in SEQ ID NO. 3;

[0011] The upstream primer N30-1610F as shown in SEQ ID NO. 4, the probe N30-1610P as shown in SEQ ID NO. 5 and the downstream primer N30-1610R as shown in SEQ ID NO. 6;

[0012] The upstream primer N30-1750F as shown in SEQ ID NO. 7, the probe N30-1750P as shown in SEQ ID NO. 8 and the downstream primer N30-1750R as shown in SEQ ID NO. 9;

[0013] The upstream primer N30-2050F as shown in SEQ ID NO. 10, the probe N30-2050P as shown in SEQ ID NO. 11 and the downstream primer N30-2050R as shown in SEQ ID NO. 12;

[0014] The upstream primer N30-4530F as shown in SEQ ID NO. 13, the probe N30-4530P as shown in SEQ ID NO. 14 and the downstream primer N30-4530R as shown in SEQ ID NO. 15;

[0015] The upstream primer N30-4920F as shown in SEQ ID NO. 16, the probe N30-4920P as shown in SEQ ID NO. 17 and the downstream primer N30-4920R as shown in SEQ ID NO. 18;

[0016] The upstream primer N30-5930F as shown in SEQ ID NO. 19, the probe N30-5930P as shown in SEQ ID NO. 20 and the downstream primer N30-5930R as shown in SEQ ID NO. 21;

[0017] The upstream primer N30-6100F as shown in SEQ ID NO. 22, the probe N30-6100P as shown in SEQ ID NO. 23 and the downstream primer N30-6100R as shown in SEQ ID NO. 24;

[0018] an upstream primer N30-7300F as set forth in SEQ ID NO. 25, a probe N30-7300P as set forth in SEQ ID NO. 26, and a downstream primer N30-7300R as set forth in SEQ ID NO. 27;

[0019] an upstream primer N30-8480F as set forth in SEQ ID NO. 28, a probe N30-8480P as set forth in SEQ ID NO. 29, and a downstream primer N30-8480R as set forth in SEQ ID NO. 30;

[0020] an upstream primer N30-12620F as set forth in SEQ ID NO. 31, a probe N30-12620P as set forth in SEQ ID NO. 32, and a downstream primer N30-12620R as set forth in SEQ ID NO. 33;

[0021] an upstream primer N30-12750F as set forth in SEQ ID NO. 34, a probe N30-12750P as set forth in SEQ ID NO. 35, and a downstream primer N30-12750R as set forth in SEQ ID NO. 36;

[0022] an upstream primer N30-13270F as set forth in SEQ ID NO. 37, a probe N30-13270P as set forth in SEQ ID NO. 38, and a downstream primer N30-13270R as set forth in SEQ ID NO. 39;

[0023] an upstream primer N30-14630F as set forth in SEQ ID NO. 40, a probe N30-14630P as set forth in SEQ ID NO. 41, and a downstream primer N30-14630R as set forth in SEQ ID NO. 42;

[0024] an upstream primer N30-14960F as set forth in SEQ ID NO. 43, a probe N30-14960P as set forth in SEQ ID NO. 44, and a downstream primer N30-14960R as set forth in SEQ ID NO. 45.

[0025] wherein the primer probe combination comprises any one or several of the primer probe combinations numbered 2-4, 6, 7, 9, 11, 13-14, 16, 20-22, 25, and 27, and the specific sequences are as follows:

[0026]

[0027]

[0028]

[0029] When single detection is performed, any one of the above primer probe combinations can be selected at random;

[0030] When double detection is performed, any two of the above 15 numbered combinations can be selected at random, wherein there are 105 combinations in any two combinations.

[0031] As a preferred, the present application finally screens out 15 groups of optimal primer probe combinations, and combines them into 7 groups of double detection combinations and 1 group of single detection combination, wherein the 7 groups of double detection combinations include: combination 1: primer probe combination (N30-1150) numbered 2 + primer probe combination (N30-5930) numbered 11; combination 3: primer probe combination (N30-1610) numbered 3 + primer probe combination (N30-4530) numbered 7; combination 4: primer probe combination (N30-13270) numbered 22 + primer probe combination (N30-4920) numbered 9; combination 5: primer probe combination (N30-7300) numbered 14 + primer probe combination (N30-14960) numbered 27; combination 6: primer probe combination (N30-6100) numbered 13 + primer probe combination (N30-8480) numbered 16; combination 7: primer probe combination (N30-12620) numbered 20 + primer probe combination (N30-12750) numbered 21; combination 8: primer probe combination (N30-1750) numbered 4 + primer probe combination (N30-14630) numbered 25); 1 group of single detection combination is combination 2: primer probe combination (N30-2050) numbered 6.

[0032] Wherein, the probe is labeled with any one of the fluorescent dyes, and the other probe must be labeled with a fluorescent dye that uses a different detection channel, and the fluorescent dye is selected from any one of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 fluorescent dyes.

[0033] Wherein, the NADC30-like PRRSV-2 includes one or more of NADC30-like PRRSV-2-SD17-36 strain, NADC30-like PRRSV-2-JSYZ24-2748 strain, NADC30-like PRRSV-2-GDCZ22-1580 strain, NADC30-like PRRSV-2-SDLY23-1742 strain, NADC30-like PRRSV-2-BJ23-2652 strain or NADC30-like PRRSV-2-HNZK24-2680 strain or other NADC30-like PRRSV-2 strains.

[0034] The concentration of the primer and the probe is 5-20 μM. Preferably, the concentration of the primer and the probe is 10 μM.

[0035] 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 NADC30-like PRRSV-2 strain.

[0036] The present application also includes a multiplex detection kit for simultaneously detecting the whole genome of NADC30-like PRRSV-2 strain, which comprises the primer-probe combination.

[0037] The multiplex detection kit further comprises other detection reagents required for multiplex PCR.

[0038] The multiplex PCR comprises real-time fluorescent quantitative PCR.

[0039] The multiplex detection kit comprises 2xPremix Ex Taq and RNase Free H2O.

[0040] The amplification procedure of the multiplex detection kit is as follows: 95℃ for 30 s, 1 cycle; 95℃ for 5 s, 60℃ for 1 min, 40 cycles.

[0041] The present application also provides a multiplex real-time fluorescent PCR method for simultaneously detecting the whole genome of NADC30-like PRRSV-2 strain using the above-mentioned composition, which comprises the following steps:

[0042] (1) designing specific primers and probe sequences for detecting the whole genome of NADC30-like PRRSV-2 strain;

[0043] (2) using different fluorescent labels for the probes for the whole genome of NADC30-like PRRSV-2 strain, each probe can be labeled with any one of the fluorescent labels, and the combination of the probe labels must be fluorescent labels that can be detected through different detection channels, which can be selected from FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5.

[0044] (3) placing the above-mentioned primers and probes for detecting the whole genome of NADC30-like PRRSV-2 strain in the same reaction tube, and using a fluorescent PCR amplifier to perform real-time fluorescent PCR reaction.

[0045] Beneficial effects: Compared with the prior art, the present application has the following advantages: compared with the prior art, the present application adopts a whole genome multi-target detection strategy, which can effectively detect and analyze the recombination of NADC30-like PRRSV-2; multiple detection is realized, and multiple gene targets can be detected simultaneously in a single reaction, which significantly improves the detection efficiency and reduces the cost of single gene detection. By designing primers and probes covering the key regions of the whole genome, the virus recombination characteristics can be accurately identified, which provides important data support for virus evolution research and prevention and control strategy making. The technology is simple to operate, the results are reliable, and it is suitable for large-scale screening of clinical samples. Specifically, the following points are included:

[0046] 1、The present application can effectively distinguish NADC30-like PRRSV-2 from other PRRSV strains by designing specific primers and probes covering the whole genome of NADC30-like PRRSV-2. The traditional single-target detection method is prone to false negative when facing virus variation or recombination, while the present application adopts a whole genome multi-target detection strategy, which greatly improves the reliability of the detection results and reduces the breeding loss caused by misjudgment.

[0047] 2、The present application realizes multiple detection and whole genome coverage of NADC30-like PRRSV-2, which can detect multiple gene targets simultaneously in one reaction, reduces the repeated use of reagents and equipment, significantly shortens the detection time, and reduces the detection cost. Through optimized primer and probe design, the detection efficiency is improved, which provides strong support for large-scale sample detection. Not only is it helpful to promote the use in the pig industry, but also provides stronger technical support for the prevention and control of the pig industry, and can be applied to epidemiological investigation and pig farm detection to quickly and accurately understand the virus variation.

[0048] 3、The present application covers multiple key gene regions of the whole genome of NADC30-like PRRSV-2, which can effectively determine and analyze the recombination of the epidemic virus to be detected. Through synchronous detection of different gene regions, the position and frequency of recombination can be accurately located, which provides important data support for virus evolution research and prevention and control. The prior art cannot effectively detect the recombination due to the limitation of target points, and the breakthrough of the present application in this aspect fills the gap of the prior art, which helps to deeply understand the variation rule of the virus and develop more effective prevention and control strategies.

[0049] 4、The application can effectively monitor the prevalence of PRRSV in pig farms, especially for the NADC30-like strain and its recombinant variants widely prevalent in current Chinese pig populations. Regular detection using the specific primer probe combination of the application can help real-time grasp the changes in viral genetic characteristics in pig populations. When the detection finds changes in the gene combination, it often indicates that the pig farm may have introduced a new recombinant strain. Given the high prevalence of the current NADC30-like strain and its complex recombinant characteristics, this multi-target detection technology can provide important technical support for the prevention and control of PRRSV in pig farms. By timely detecting strain variation, it can guide pig farms to take targeted immunization and management measures to effectively control the spread and prevalence of PRRSV. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is an example of a specific conserved region screening design of the full genome of NADC30-like PRRSV-2;

[0051] Figure 2A is an example of good specificity of the full genome multi-target detection method of NADC30-like PRRSV-2;

[0052] Figure 2B is an example of poor specificity of the full genome multi-target detection method of NADC30-like PRRSV-2;

[0053] Figure 3A is a test result chart of the full genome multi-target multiplex detection method of NADC30-like PRRSV-2 for non-recombinant strain (SD17-36) detection performance;

[0054] Figure 3B is a test result chart of the full genome multi-target detection method of NADC30-like PRRSV-2 for recombinant strain (JSTZ24-2748) detection performance;

[0055] Figure 3C is a recombinant analysis result chart of SD17-36 strain and JSTZ24-2748 strain;

[0056] Figure 4A is an example of inhibitory combination of the full genome multi-target multiplex detection method of NADC30-like PRRSV-2;

[0057] Figure 4B is an example of amplification enhancement combination of the full genome multi-target multiplex detection method of NADC30-like PRRSV-2;

[0058] Figure 4CFigure 6 is a full genome multi-target multiplex detection method V2 kit test result graph of NADC30-like PRRSV-2.

[0059] Figure 5 is a full genome multi-target multiplex detection method V1 kit test result graph of NADC30-like PRRSV-2.

[0060] Figure 6 is a full genome multi-target multiplex detection method V2 kit test result graph of NADC30-like PRRSV-2.

[0061] Figure 7 is a full genome multi-target multiplex detection method V3 kit test result graph of NADC30-like PRRSV-2. DETAILED DESCRIPTION

[0062] The embodiments of the present application will be described in detail below with reference to examples. The following examples are only used to illustrate the present application, but not to limit the scope of the present application. Only modifications or replacements of the method, steps or conditions of the present application without changing the essence or deviating from the spirit of the present application, still belong to the protection scope of the present application.

[0063] The conventional experimental methods in the following examples refer to the "Molecular Cloning Experiment Guide" third edition (Beijing: Science Press, 2002) edited by Sambrook et al., and the use of instruments refers to the instrument operation manual. If not specifically indicated, 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: HP-PRRSV-2 XJ17-5 (GenBank accession number: MK759853) strain, NADC30-like PRRSV-2 (SD17-36 (MH121061) strain / JSYZ24-2748 (PX148102) strain / GDCZ22-1580 (PX148098) strain / SDLY23-1742 (PQ373813) strain / BJ23-2652 (PX148100) strain / HNZK24-2680 (PX148101) strain), VR2332-like PRRSV-2 JSYC-2005-2 (MT746146) strain and PRRSV-1 AHEU2024-2671 (PQ640355) strain are isolated and preserved by the laboratory. NADC34-like PRRSV-2 rBJ1805-2 (PQ373814) strain (non-recombinant strain preservation number CCTCC NO: V202250, patent application number: 202210804949.1).

[0064] In the embodiments of the present application, other reagents used are: RNase Free H2O purchased from Solarbio Company; 2xPremix Ex Taq purchased from TAKARA Company; QIAGEN RNAeasy Mini Kit purchased from KANGJIE BIOENGINEERING CO., LTD; HiScript III 1st Strand cDNA Synthesis Kit purchased from NOVGENE BIOLOGICAL TECHNOLOGIES CO., LTD; DNA Marker purchased from ZHEJIANG BOERJIN BIOTECHNOLOGY CO., LTD. The primers used in the experiment were synthesized by Suzhou JUNWENZHI Biological Technology Co., Ltd. and General Biological (Anhui) Co., Ltd., and the probes were synthesized by Kunshan Punopu Biological Technology Co., Ltd. and General Biological (Anhui) Co., Ltd.

[0065] Establishment of whole genome multi-target multiplex detection method for NADC30-like PRRSV-2

[0066] 1. Design and screening of primers and probes for whole genome detection of NADC30-like PRRSV-2

[0067] 100 complete genome sequences covering all major lineages of PRRSV-1 and PRRSV-2 were downloaded from the GenBank database, and multiple sequence alignment analysis was performed using sequence alignment software DNAMAN. Based on the highly conserved and different subtype-specific gene regions in PRRSV strains (such as Figure 1 ), the selected regions on the whole genome include 50bp-500bp (No. 1), 1000bp-2300bp (No. 2-No. 6), 4400bp-6500bp (No. 7-No. 13), 7000bp-9000bp (No. 14-No. 16), 9500bp-11000bp (No. 17-No. 18), 11500bp-14000bp (No. 19-No. 24), 14300bp-15050bp (No. 25-No. 27).

[0068] According to the above relatively conservative region design NADC30-like PRRSV-2 full gene coverage specific detection primer and probe. The 5' end of the probe is labeled with different fluorescent groups (FAM and HEX) respectively. The specific 27 group primer probe sequence is shown in Table 1. The NADC30-like PRRSV-2 (SD17-36 strain) RNA extracted by QIAGEN RNAeasy Mini Kit was used as template, and the HiScript III 1st Strand cDNA Synthesis Kit was used for reverse transcription to cDNA, and then the reaction system shown in Table 2 and the reaction condition shown in Table 3 were used for qPCR amplification and signal collection. Eliminate 5 groups of primer-probe combinations without amplification signal, and retain 22 groups of effective primer-probe combinations for subsequent experiments. The specific primer probe sequence is shown in Table 1 (number 1-4, 6-9, 11-17, 20-22, 24-27).

[0069] Table 1 NADC30-like PRRSV-2 multiplex detection primer probe table

[0070]

[0071]

[0072]

[0073] Table 2 NADC30-like PRRSV-2 multiplex detection reaction system

[0074]

[0075] Table 3 NADC30-like PRRSV-2 multiplex detection amplification program

[0076]

[0077] 2、Specificity test

[0078] The RNA of different PRRSV strains (NADC34-like PRRSV-2rBJ1805-2 strain, NADC30-like PRRSV-2SD17-36 strain, HP-PRRSV-2XJ17-5 strain, VR2332-like PRRSV-2JSYC-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. The specificity of the 22 selected primer probe combinations was systematically evaluated during the detection of different PRRSV strains using real-time fluorescent PCR method. The specific amplification conditions and amplification system are shown in Step 1. The results show that some primer probe combinations (Table 1, numbered as 2-4, 6-9, 11, 13-14, 16, 20-22, 25, 27) can achieve specific amplification of specific strains. For example, as shown in Figure 2A , the N30-8480 group only produces specific amplification signals for the NADC30-like SD17-36 strain during detection, and no obvious cross-reaction occurs for other strains, indicating that it has high specificity and can accurately identify the target strain. It can be used as a powerful tool for specific detection of NADC30-like strains in subsequent experiments.

[0079] However, not all primer probe combinations have ideal specificity. For example, as shown in Figure 2B , the N30-9720 group has obvious non-specific amplification during detection. This combination produces amplification signals for strains of different lineages, indicating that it cannot effectively distinguish between target strains and other strains, and has poor specificity. This non-specific amplification can lead to misjudgment of the detection results and affect the accurate identification and analysis of PRRSV strains. In order to ensure the accuracy and reliability of the experiment, all combinations with non-specific amplification were discarded, and 16 primer probe combinations with high specificity were finally selected for subsequent experiments. The specific primer probe sequences are shown in Table 1 (numbered as 2-4, 6-9, 11, 13-14, 16, 20-22, 25, 27).

[0080] 3. Recombinant strain detection

[0081] To verify the accuracy and reliability of the screened primer probe combinations in detecting NADC30-like PRRSV recombinant strains, NADC30-like recombinant strain JSYZ24-2748 and NADC30-like non-recombinant strain SD17-36 were selected as test objects, and the 16 groups of high-specificity primer probe combinations (numbered 2-4, 6-9, 11, 13-14, 16, 20-22, 25, and 27) finally screened were systematically tested. In the test, real-time fluorescent PCR technology was used to quantitatively analyze the 16 groups of primer probe combinations, and the specific amplification conditions and amplification system are shown in Step 1, to evaluate their performance in detecting recombinant strains. As shown in Figure 3A , the quantitative amplification curve and statistical analysis results clearly show that all primer probe combinations can effectively amplify the SD17-36 strain, which indicates that these combinations have high sensitivity and stability in detecting this strain. However, for the JSYZ24-2748 strain, Figure 3B , the results show that only some primer probe combinations can produce amplification signals, while the remaining combinations fail to detect obvious amplification products. This difference indicates that different primer probe combinations exhibit significant specificity differences in detecting different strains.

[0082] To further verify the ability of these primer probe combinations to distinguish between recombinant strains and non-recombinant strains, RDP4 and Simplot software were used to perform detailed recombinant analysis on the SD17-36 strain and the JSYZ24-2748 strain. After comparing the results of the recombinant analysis with the detection results of the quantitative PCR, it was found that they were highly consistent (as shown in Figure 3C ). This result indicates that the primer probe combinations not only can accurately detect the presence of PRRSV strains, but also can effectively distinguish between recombinant strains and non-recombinant strains, which is of great significance for the accurate detection and classification of PRRSV, as well as the in-depth study of the genetic variation and epidemic trend of PRRSV.

[0083] 4. Probe labeling and combination testing

[0084] After labeling the primer probe combinations with different channels, the primer probe combinations were optimized. Based on the rich experience accumulated in the field of primer probe design for more than ten years, the present application has fully considered and as far as possible avoided most potential factors that may interfere with multiplex differential detection when designing primer probe combinations. These factors include but are not limited to the possible formation of dimers between primer probe sequences, the mutual interference between primer probe combinations of different targets, and the difference in annealing temperature, etc. However, despite careful consideration and optimization during the design stage, many challenges were still encountered during the actual test process. For example, in the process of Figure 4AThe results of single-base detection using two primer and probe sets, N30-1150 and N30-8480, are shown in the image (e.g., Figure 4A (a and b in the text) and the detection results when they are combined for dual detection (e.g.) Figure 4A (c) From the single-detection results, the N30-8480 group can effectively amplify SDLY23-1742 and BJ23-2652 strains. However, when N30-1150 (primer-probe combination number 2) and N30-8480 (primer-probe combination number 16) are combined for dual detection, the amplification ability of the N30-8480 group for these two strains disappears. This phenomenon may be related to the mutual inhibition between the primers and probes.

[0085] Furthermore, contrary to the aforementioned mutual inhibition phenomenon, combining individual primers and probes can actually increase the number of amplifiable strains. For example... Figure 4B As shown, primer-probe combination N30-12620 (numbered 20) and primer-probe combination N30-12750 (numbered 21) are used in single detection (e.g.) Figure 4B In the samples a and b), group N30-12620 showed no amplification signal against either GDCZ22-1580 or HNZK24-2680 strains. However, when these two primers and probes were combined (e.g., ...), ... Figure 4B In group c), the N30-12620 group was able to successfully amplify both strains. This phenomenon may be related to the synergistic effect between the primers and probes.

[0086] To complicate matters further, some primer-probe combinations are incompatible with any other primer-probe combination. For example... Figure 4C As shown, the N30-1750 group (primer-probe combination number 4) and the N30-2050 group (primer-probe combination number 6) in single detection (e.g.) Figure 4C Both a and b in the above can amplify their respective target strains normally, but when they are combined (e.g., ... Figure 4C In section c), the N30-2050 group showed no amplification signal for any strain. To further verify this incompatibility, the N30-2050 group was tested with primer-probe combinations from all other different channels. The results showed that this group showed no amplification signal in any combination. 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-probe sequences, preventing the primers and probes from binding properly to the target template during the reaction, thus failing to initiate the amplification reaction.

[0087] After repeated testing and verification, the incompatible primer probe was finally retained for single detection to ensure the accuracy and reliability of the detection results. In order to further optimize the primer probe combination, on the basis of the real-time fluorescent RT-PCR detection method, considering the cost of using the kit and the position of the primer probe, etc., the primer probe combination was strictly screened and optimized. Through careful comparison and analysis of the amplification efficiency, sensitivity and other key parameters of different combinations, 15 groups of optimal primer probe combinations were finally selected, and they were combined into 7 groups of duplex detection combinations (combination 1: primer probe combination No. 2 + primer probe combination No. 11; combination 3: primer probe combination No. 3 + primer probe combination No. 7; combination 4: primer probe combination No. 4 + primer probe combination No. 9; combination 5: primer probe combination No. 14 + primer probe combination No. 27; combination 6: primer probe combination No. 13 + primer probe combination No. 16; combination 7: primer probe combination No. 20 + primer probe combination No. 21; combination 8: primer probe combination No. 22 (N30-13270) + primer probe combination No. 25 (N30-14630)) + 1 group of single detection combination (combination 2: primer probe combination No. 6) (in which the primer probe No. 8 (N30-4750) was removed) for the development of the kit (Figures 5-7).

[0088] 5. Kit development and iteration

[0089] After the optimization of the primer probe combination, it was assembled into a kit for actual application testing. The test kit at least includes the main components: negative control, positive control, nuclease-free water, RT-PCR reaction solution, reverse transcriptase and Taq enzyme mixed solution, 15 groups of primer probe single mixed solution or duplex mixed solution, etc. However, some problems were found during the test. For example, the V1 kit developed, 15 groups of primer probe pairing combinations were formed into 7 groups of duplex detection combinations and 1 group of single detection combination (see Figures 5-7 for specific combination methods), and after assembly, a comprehensive test was carried out.

[0090] Specifically, the N30-13270P-HEX group in the duplex combination 8 (No. 22 (N30-13270) + No. 25 (N30-14630)) in the V1 kit did not appear after assembly. The phenomenon of amplification, which is clearly shown in Figure 5A However, before assembly, this combination can successfully amplify the target template Figure 5B), indicating that the problem may have occurred during assembly or other components of the kit affected the primer probe. This difference in performance before and after formed a sharp contrast, which attracted our high attention. In order to solve this problem, various methods were tried. First, considering that the quality of primer probe synthesis may be the root of the problem, the V2 kit was re-synthesized by Universal Biological (Anhui) Co., Ltd. all primers-probes, instead of the original supplier Suzhou Jinyuzhi Biological Technology Co., Ltd. and Kunshan Punopu Biological Technology Co., Ltd. However, the comparison of test results before and after assembly showed that the problem was still not solved( Figure 6A and Figure 6B ), which indicated that the problem may not be caused entirely by the quality of synthesis, but may be related to other factors of the kit. After further analysis and adjustment, the duplex combination 8 was optimized in V3 kit, adjusted to No. 4 (N30-1750) + No. 25 (N30-14630), while the duplex combination 4 was adjusted to No. 9 (N30-4920) + No. 22 (N30-13270). After this adjustment, the test results showed the expected( Figure 7A and Figure 7B ), indicating that the new combination could amplify normally, solving the previous problem. This result also further verified the importance of primer probe combination optimization for the performance of the kit.

[0091] Through this series of tests and adjustments, the primer probe combination scheme of V3 kit was finally determined, ensuring the accuracy and reliability of the kit in actual application. This process not only solved the problem, but also provided valuable experience and reference for the production and application of subsequent kits.

Claims

1. A primer probe combination for NADC30-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 N30-1150F as shown in SEQ ID NO. 1, a probe N30-1150P as shown in SEQ ID NO. 2, and a downstream primer N30-1150R as shown in SEQ ID NO. 3; an upstream primer N30-1610F as shown in SEQ ID NO. 4, a probe N30-1610P as shown in SEQ ID NO. 5, and a downstream primer N30-1610R as shown in SEQ ID NO. 6; an upstream primer N30-1750F as shown in SEQ ID NO. 7, a probe N30-1750P as shown in SEQ ID NO. 8, and a downstream primer N30-1750R as shown in SEQ ID NO. 9; an upstream primer N30-2050F as shown in SEQ ID NO. 10, a probe N30-2050P as shown in SEQ ID NO. 11, and a downstream primer N30-2050R as shown in SEQ ID NO. 12; an upstream primer N30-4530F as shown in SEQ ID NO. 13, a probe N30-4530P as shown in SEQ ID NO. 14, and a downstream primer N30-4530R as shown in SEQ ID NO. 15; an upstream primer N30-4920F as shown in SEQ ID NO. 16, a probe N30-4920P as shown in SEQ ID NO. 17, and a downstream primer N30-4920R as shown in SEQ ID NO. 18; an upstream primer N30-5930F as shown in SEQ ID NO. 19, a probe N30-5930P as shown in SEQ ID NO. 20, and a downstream primer N30-5930R as shown in SEQ ID NO. 21; an upstream primer N30-6100F as shown in SEQ ID NO. 22, a probe N30-6100P as shown in SEQ ID NO. 23, and a downstream primer N30-6100R as shown in SEQ ID NO. 24; an upstream primer N30-7300F as shown in SEQ ID NO. 25, a probe N30-7300P as shown in SEQ ID NO. 26, and a downstream primer N30-7300R as shown in SEQ ID NO. 27; an upstream primer N30-8480F as shown in SEQ ID NO. 28, a probe N30-8480P as shown in SEQ ID NO. 29, and a downstream primer N30-8480R as shown in SEQ ID NO. 30; an upstream primer N30-12620F as shown in SEQ ID NO. 31, a probe N30-12620P as shown in SEQ ID NO. 32, and a downstream primer N30-12620R as shown in SEQ ID NO. 33; an upstream primer N30-12750F as set forth in SEQ ID NO. 34, a probe N30-12750P as set forth in SEQ ID NO. 35, and a downstream primer N30-12750R as set forth in SEQ ID NO. 36; an upstream primer N30-13270F as set forth in SEQ ID NO. 37, a probe N30-13270P as set forth in SEQ ID NO. 38, and a downstream primer N30-13270R as set forth in SEQ ID NO. 39; an upstream primer N30-14630F as set forth in SEQ ID NO. 40, a probe N30-14630P as set forth in SEQ ID NO. 41, and a downstream primer N30-14630R as set forth in SEQ ID NO. 42; an upstream primer N30-14960F as set forth in SEQ ID NO. 43, a probe N30-14960P as set forth in SEQ ID NO. 44, and a downstream primer N30-14960R as set forth in SEQ ID NO.

45.

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 The NADC30-like PRRSV-2 includes one or more of NADC30-like PRRSV-2-SD17-36 strain, NADC30-like PRRSV-2-JSYZ24-2748 strain, NADC30-like PRRSV-2-GDCZ22-1580 strain, NADC30-like PRRSV-2-SDLY23-1742 strain, NADC30-like PRRSV-2-BJ23-2652 strain, NADC30-like PRRSV-2-HNZK24-2680 strain or other NADC30-like PRRSV-2 strains.

4. The primer probe combination of claim 1, wherein The concentrations of the primers and probes are all 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 NADC30-like PRRSV-2 strains.

6. A multiplex detection kit capable of simultaneously detecting the whole genome of NADC30-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 30s for 1 cycle; 95℃ for 5s, 60℃ for 1min for 40 cycles.

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