Multiplex fluorescent quantitative primer and probe combination for differentiating porcine reproductive and respiratory syndrome virus variants and its application

Abstract

The application provides a multiplex fluorescent quantitative primer and probe combination for differentiating and diagnosing porcine reproductive and respiratory syndrome virus (PRRSV) strains and application thereof, including three groups of primers and probes for PRRSV, NADC30 and NADC34 respectively; further including primers and probes for 18S rRNA sequence as internal standards in detection. Further provided is a multiplex fluorescent quantitative RT-PCR kit for differentiating and diagnosing typical or atypical respiratory and reproductive disorders of pigs caused by different types of PRRSV strains, including a reaction solution packaged by the kit, and each primer and corresponding probe group is packaged or mixedly packaged. The application can simultaneously differentiate and diagnose various high-incidence pathogenic strains (PRRSV, NADC30 and NADC34) of porcine reproductive and respiratory syndrome through design, optimization and screening of primers and probes, and the amplification of the three target genes does not interfere with each other, and the application has high specificity, high sensitivity, good repeatability, can accurately diagnose viral infection and has high application value and huge economic benefits.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and veterinary medicine, and in particular relates to multiplex fluorescent quantitative primer and probe combinations for diagnosing variants of porcine reproductive and respiratory syndrome and their applications. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), commonly known as "blue ear disease" virus, is a serious infectious disease that severely threatens the global pig industry. Pregnant sows infected with PRRSV mainly exhibit symptoms such as fever, lethargy, and drowsiness; late-pregnancy abortion, stillbirth, and mummified fetuses; delayed estrus; and low conception rates. Piglets suffer from high fever, respiratory distress, bluish or reddish discoloration of some ears, and high mortality. PRRSV has immunosuppressive effects and is prone to secondary infections, leading to complex disease progression. Traditional live vaccines are increasingly ineffective in controlling the disease, making prevention and control challenging. Therefore, effective control requires a highly sensitive and accurate detection system to precisely cut off the source of infection and transmission routes of PRRSV.

[0003] PRRSV belongs to the order Nidovirales, family Arteriviridae, and genus Arteriviridae. It is an enveloped, single-stranded, positive-sense RNA virus. The virus particle is spherical and has a surface similar to that of coronaviruses with protrusions. The PRRSV genome contains at least 10 open reading frames (ORFs): ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5, ORF6, and ORF7 (Deng Y, Pan Y, Wang D, et al. Complete genome sequence of porcine reproductive and respiratory syndrome virus strain QY2010 reveals a novel subgroup emerging in China. J Virol. 2012; 86(14):7719-7720.). PRRSV has two main genotypes, the European type and the American type, which share approximately 60% nucleotide identity (Allende R, Lewis TL, Lu Z, et al. North American and European porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions. J Gen Virol. 1999; 80(Pt 2):307-315.). ORFs 1a and 1b encode non-structural proteins (NSPs), with the Nsp2 gene being the largest NSP. The gene encoding this protein is prone to base deletions, mutations, and insertions, resulting in variations in length among different viral strains. The extremely high variability of the NSP2 gene makes it an important indicator for monitoring PRRSV variation. PRRSV adapts to relatively humid and cool environments, maintaining its infectivity for extended periods at lower temperatures and pH 6.5–7.5. PRRSV infects pigs of all breeds and ages, primarily harming sows and piglets. Infected and carrier pigs are the main sources of PRRSV infection. The virus is present in the feces, secretions, and urine of infected sows. PRRSV is transmitted through contact, air, and semen, and can also be transmitted vertically through the placenta. PRRSV was first reported in Europe and the United States in the early 1990s. In my country, PRRSV was first reported in 1996; in 2006, a new PRRSV characterized by high morbidity and mortality emerged, named highly pathogenic PRRSV (HP-PRRSV).The NADC30-like PRRSV strain was first discovered in 2013. This novel strain spread rapidly and has become one of the main prevalent strains in my country, causing huge economic losses to the pig industry (Li C, 2016, Li C, Zhuang J, Wang J, et al. Outbreak Investigation of NADC30-Like PRRSV in South-East China. Transbound Emerg Dis. 2016; 63(5):474-479.). Compared with the classic strain, the NADC30-like PRRSV strain leads to increased mortality in piglets, is prone to secondary infections of other diseases, and can cause persistent infection. The first case of NADC34-like PRRSV in China was reported in 2018 (Zhang HL, 2018). Compared to the currently prevalent NADC30-like PRRSV strains, NADC34-like PRRSV has the potential to become a local strain in China (Xu H, Li C, Li W, et al. Novel characteristics of Chinese NADC34-like PRRSV during 2020-2021. Transbound Emerg Dis. 2022 Feb 19.). Due to its high mutation rate, PRRSV has led to the emergence of many new and complex circulating strains, and cases of mixed infections with multiple strains exist, posing a serious challenge to the prevention and control of this disease. Therefore, a highly specific and sensitive diagnostic method is needed to differentiate between different types of PRRSV.

[0004] Clinical methods for detecting PRRSV include virus isolation and identification, enzyme-linked immunosorbent assay (ELISA), immunoperoxidase monolayer assay, immunofluorescence, polymerase chain reaction (PCR), gene chips, and quantitative real-time PCR (qPCR). Among these, TaqMan probe-based multiplex qPCR offers advantages such as high specificity, high sensitivity, accuracy, and quantification. Therefore, multiplex qPCR is becoming increasingly indispensable for RNA pathogen detection. Although more and more multiplex qPCR methods are available, this method also faces two challenges: First, it is difficult to develop a stable, efficient, and specific primer-probe combination. This combination must be able to simultaneously amplify multiple target genes in the same system, ensure the specificity and efficiency of each primer pair, and prevent primer dimers from forming and affecting the reaction system. Furthermore, it is necessary to fully optimize primer concentration ratios, annealing temperatures, and other reaction conditions. Second, multiplex qPCR requires a high level of professional expertise. Operators lacking sufficient basic skills are prone to inaccurate results such as false positives and false negatives, and are also susceptible to environmental and sample contamination.

[0005] POCT (point-of-care testing) is a microfluidic technology and one of the leading technologies in international gene testing (Vashist SK. Point-of-Care Diagnostics: Recent Advances and Trends. Biosensors (Basel). 2017, 7(4). pii: E62.). This detection method integrates nucleic acid extraction with quantitative fluorescence gene detection, combined with microfluidic technology, to achieve the goal of gene detection with the sample entering and the result exiting. The entire reaction process is completely closed, with no aerosol release, and the detection time is shortened to about 1 hour, which reduces manual operation time and avoids result errors caused by human experimental operation mistakes. To date, international companies such as Cepheid and Roche have launched such POCT products. Their high efficiency, completely closed and pollution-free characteristics, and lack of the need for professional personnel have made them very popular in Europe and the United States. There are relatively few products on the market in this field in my country, but research and development is ongoing. Summary of the Invention

[0006] To date, there are several areas for improvement in the detection of porcine PRRSV: First, the viral RNA extraction step and the molecular reaction stage are separated. During RNA extraction, improper operation can easily lead to RNA degradation and false results. Second, in the molecular reaction stage, inadequate hardware or human error can cause aerosol contamination, resulting in false positives. These factors make it impossible to determine the reliability of experimental results and miss the optimal time for intervention. In summary, this invention addresses these two major pain points of multiplex quantitative RT-PCR by performing sequence analysis on three PRRSV strains (sequences 13-15): the conserved gene fragment, the NADC30 variant, and the NADC34 variant. A set of amplification primers and corresponding probes for these three strains were designed for the detection of porcine reproductive and respiratory syndrome (PRRS). The reaction is carried out in the same system, ensuring that the starting sample size for target gene amplification is the same, avoiding experimental errors caused by batch operations. In cases of mixed infection, the pathogen type can be accurately identified, which improves work efficiency and reduces labor costs. The multiplex quantitative RT-PCR method in this invention has a sensitivity 10 to 100 times that of traditional RT-PCR. It can simultaneously and specifically amplify multiple pathogens in one reaction tube, which not only ensures the accuracy and timeliness of diagnostic results, but also improves detection efficiency and reduces labor costs, making it extremely cost-effective.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In the first aspect, a primer pair and probe combination for the differential diagnosis of at least one of PRRSV / NADC30 / NADC34 is provided, comprising:

[0009] (1) Primer pairs and probes for the PRRSV N gene, the primer pairs are shown in Sequence 1 and Sequence 2, and the probe sequence is shown in Sequence 3;

[0010] (2) Primer pairs and probes for the NADC30 Nsp2 gene are shown in sequences 4 and 5, and probe sequences are shown in sequence 6.

[0011] (3) Primer pairs and probes for the NADC34 Nsp2 gene are shown in Sequence 7 and Sequence 8, and the probe sequence is shown in Sequence 9.

[0012] Preferably, the two ends of the probe are modified with fluorescent labels, wherein the 5' end of sequence 3 is modified with FAM and the 3' end is modified with BHQ1; the 5' end of sequence 6 is modified with HEX and the 3' end is modified with BHQ1; and the 5' end of sequence 9 is modified with CY5 and the 3' end is modified with BHQ3.

[0013] In any of the above schemes, it is preferable to use 18S rRNA as an internal standard and design internal standard primers and probes to identify PRRSV, NADC30, and NADC34.

[0014] In any of the above schemes, the internal standard primer pair sequences are shown in Sequence 10 and Sequence 11, respectively, and the probe sequence is shown in Sequence 12; and the two ends of the internal standard probe are fluorescently labeled, with the 5' end modified with ROX and the 3' end modified with BHQ2.

[0015] Secondly, the present invention further provides a kit for the differential diagnosis of at least one of PRRSV / NADC30 / NADC34, comprising a combination of target detection primers and probes as described in items (1) to (3) above, and a combination of primers and probes for an internal standard. In a specific embodiment, each primer pair and corresponding probe are packaged separately or mixed together.

[0016] Preferably, the primer pairs and probes in the kit are embedded in a premixed solution containing 2.5 mM Mn2+; the concentrations of the primers and probes are as follows: 0.3 μM each for primers shown in sequence 1 and sequence 2, and 0.2 μM for probe shown in sequence 3; 0.3 μM each for primers shown in sequence 4 and sequence 5, and 0.2 μM for probe shown in sequence 6; 0.3 μM each for primers shown in sequence 7 and sequence 8, and 0.2 μM for probe shown in sequence 9; 0.2 μM each for primers shown in sequence 10 and sequence 11, and 0.15 μM for probe shown in sequence 12.

[0017] Preferably, any of the above embodiments further includes a lysis extract and a sample washing buffer. Specifically, the main components of the lysis extract are: 3M Gu.HCl, 0.8M NaCl, 30% isopropanol, 3% Tween 20, 1% Triton-X10, 0.3mM EDTA, and 12mM Tris.HCl. The sample washing buffer can be a washing buffer conventionally used in the art.

[0018] In specific implementation methods, the detection of the present invention can target one or more of PRRSV, NADC30, and NADC34, that is, to achieve simultaneous detection of one, two, or three targets, thereby enabling the identification of porcine reproductive and respiratory syndrome (PRRS).

[0019] The primer pairs and probes in this invention, used for the identification and diagnosis of porcine reproductive and respiratory syndrome (PRRS), can be applied, but are not limited to, to the detection kit described in the applicant's previous Chinese patent application 202010147432.0, to achieve integrated MAOPA detection. The detection kit described in that patent application has multiple separation chambers, with plungers blocking adjacent chambers. Each separation chamber contains lysis buffer, washing buffer, and reaction solution. During the detection reaction, each plunger is pushed, opening the separation chambers. The instrument then uses electromagnetic control to guide the magnetic beads carrying nucleic acids through each separation chamber. After sample lysis, the magnetic beads adsorb nucleic acids for washing and reaction. Finally, the instrument captures fluorescence from the reaction solution externally.

[0020] Thirdly, the present invention also provides a detection kit for the differential diagnosis of at least one of PRRSV / NADC30 / NADC34, the structure of which is that the kit has four separate chambers, the walls of which are similar to the partitions of a conventional molecular laboratory, wherein the first chamber is used for sample lysis and nucleic acid extraction; the second chamber is used to embed sample washing solution I for the first washing of nucleic acid; the third chamber is used to embed sample washing solution II for the second washing of nucleic acid; and the fourth chamber is used to embed reaction reagents for the multiplex quantitative PCR reaction stage.

[0021] Preferably, the chambers are blocked by plungers, and the separation chambers are connected. The instrument carries the sample nucleic acid through each separation chamber sequentially via magnetic beads in the electromagnetic control cartridge.

[0022] The advantages of this invention are: It achieves differential diagnosis of PRRSV, NADC30, and NADC34 in a single reaction, confirming both the presence and severity of porcine reproductive and respiratory syndrome virus (PRRSV) infection and identifying single, dual, or multiple infections, making it more efficient and time-saving than single-gene detection. By analyzing the selected amplification targets—PRRSV N gene, NADC30 Nsp2 gene, and NADC34 Nsp2 gene sequences—optimal primers are designed, optimized, and screened, resulting in independent amplification of the three targets with good sensitivity, specificity, and repeatability. It is also suitable for a two-step reaction combining annealing and extension, offering significant advantages over existing technologies. Therefore, the kit developed based on this invention has excellent application prospects. Furthermore, the PRRSV, NADC30, and NADC34 multiplex real-time PCR kit of this invention can be applied to integrated MAOPA detection, truly achieving a fully enclosed, pollution-free, integrated reaction from "sample in" to "result out." Manually adding the sample to the cartridge is sufficient; all other steps are automated, greatly reducing labor and time costs, minimizing human error, and avoiding subjective result judgment. It does not generate aerosol pollution and also ensures the health of testing personnel. Attached Figure Description

[0023] Figure 1 PRRSV standard amplification curve, where 1.1 × 10 7 pUC57-PRRSV-N plasmid; 2.1 × 10 6 pUC57-PRRSV-N plasmid; 3.1 × 10 5 pUC57-PRRSV-N plasmid; 4.1 × 10 4 pUC57-PRRSV-N plasmid; 5.1 × 10 3 pUC57-PRRSV-N plasmid; 6.1 × 10 2 pUC57-PRRSV-N plasmid; 7.1 × 10 1 pUC57-PRRSV-N plasmid; 8.1 × 10 0 pUC57-PRRSV-N plasmid; 9.H2O;

[0024] Figure 2 Amplification curves of NADC30 standard, where 1.1 × 10⁻⁶ 7 pUC57-NADC30-Nsp2 plasmid; 2.1 × 10 6 pUC57-NADC30-Nsp2 plasmid; 3.1×10 5 pUC57-NADC30-Nsp2 plasmid; 4.1×10 4 pUC57-NADC30-Nsp2 plasmid; 5.1 × 10 3 pUC57-NADC30-Nsp2 plasmid; 6.1×10 2 pUC57-NADC30-Nsp2 plasmid; 7.1 × 10 1 pUC57-NADC30-Nsp2 plasmid; 8.1×10 0 pUC57-NADC30-Nsp2 plasmid; 9.H2O;

[0025] Figure 3 Amplification curve of NADC34 standard, where 1.1×10 7 pUC57-NADC34-Nsp2 plasmid; 2.1 × 10 6 pUC57-NADC34-Nsp2 plasmid; 3.1×10 5 pUC57-NADC34-Nsp2 plasmid; 4.1×10 4 pUC57-NADC34-Nsp2 plasmid; 5.1 × 10 3 pUC57-NADC34-Nsp2 plasmid; 6.1×10 2 pUC57-NADC34-Nsp2 plasmid; 7.1 × 101 pUC57-NADC34-Nsp2 plasmid; 8.1×10 0 pUC57-NADC34-Nsp2 plasmid; 9.H2O;

[0026] Figure 4 The specificity of PRRSV / NADC30 / NADC34 multiplex real-time quantitative PCR was determined by the following components: 1. pUC57-PRRSV-N plasmid; 2. pUC57-NADC30-Nsp2 plasmid; 3. pUC57-NADC34-Nsp2 plasmid; 4. pUC57-18S-Nsp2 plasmid; 5. CSFV RNA; 6. TGEV RNA; 7. PDCoV RNA; 8. PCV2 DNA; 9. PCV3 RNA; 10. PPV DNA; 11. H2O.

[0027] Figure 5 The sensitivity of PRRSV / NADC30 / NADC34 multiplex quantitative PCR, where 1-9.1×10 5 pUC57-PRRSV-N plasmid; 1×10 5 pUC57-NADC30-Nsp2 plasmid; 1×10 5 pUC57-NADC34-Nsp2 plasmid; 10⁻¹⁸.1 × 10⁻¹⁰ 1 pUC57-PRRSV-N plasmid; 1×10 1 pUC57-NADC30-Nsp2 plasmid; 1×10 1 pUC57-NADC34-Nsp2 plasmid; 19.H2O;

[0028] Figure 6 Intra-batch replicates of PRRSV / NADC30 / NADC34 multiplex real-time quantitative PCR, of which 1-9.1×10 6 pUC57-PRRSV-N plasmid; 1×10 6 pUC57-NADC30-Nsp2 plasmid; 1×10 6 pUC57-NADC34-Nsp2 plasmid; 10⁻¹⁸.1 × 10⁻¹⁰ 5 pUC57-PRRSV-N plasmid; 1×10 5 pUC57-NADC30-Nsp2 plasmid; 1×10 5 pUC57-NADC34-Nsp2 plasmid; 19-27.1×10 3 pUC57-PRRSV-N plasmid; 1×10 3 pUC57-NADC30-Nsp2 plasmid; 1×103 pUC57-NADC34-Nsp2 plasmid; 28-30.H2O;

[0029] Figure 7 Inter-batch replicates of PRRSV / NADC30 / NADC34 multiplex real-time quantitative PCR, wherein: 1-3.1×10⁶ pUC57-PRRSV-Nsp2 plasmid; 1×10⁶ pUC57-NADC30-Nsp2 plasmid; 1×10⁶ pUC57-NADC34-Nsp2 plasmid; 4-6.1×10⁵ pUC57-PRRSV-Nsp2 plasmid; 1×10⁵ pUC57-NADC30-Nsp2 plasmid; 1×10⁵ pUC57-NADC34-Nsp2 plasmid; 7-9.1×10³ pUC57-PRRSV-Nsp2 plasmid; 1×10³ pUC57-NADC30-Nsp2 plasmid; 1×10³ pUC57-NADC34-Nsp2 plasmid; 10. H₂O;

[0030] Figure 8 Inter-batch replicates of PRRSV / NADC30 / NADC34 multiplex real-time quantitative PCR. The contents are as follows: 1-3.1×10⁶ pUC57-PRRSV-Nsp₂ plasmid; 1×10⁶ pUC57-NADC30-Nsp₂ plasmid; 1×10⁶ pUC57-NADC34-Nsp₂ plasmid; 4-6.1×10⁵ pUC57-PRRSV-Nsp₂ plasmid; 1×10⁵ pUC57-NADC30-Nsp₂ plasmid; 1×10⁵ pUC57-NADC34-Nsp₂ plasmid; 7-9.1×10³ pUC57-PRRSV-Nsp₂ plasmid; 1×10³ pUC57-NADC30-Nsp₂ plasmid; 1×10³ pUC57-NADC34-Nsp₂ plasmid; 10. H₂O;

[0031] Figure 9 The PRRSV / NADC30 / NADC34 multiplex real-time quantitative PCR kit is used for integrated detection. It includes: 1. Internal standard; 2. pUC57-PRRSV-N plasmid; 3. pUC57-NADC30-Nsp2 plasmid; 4. pUC57-NADC34-Nsp2 plasmid. Detailed Implementation

[0032] To enable those skilled in the art to better understand the solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the examples of the present invention. Obviously, the described examples are only a part of the examples of the present invention, and not all of the examples. Based on the examples in the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] Example 1

[0034] I. Preparation of plasmid standards

[0035] High recombination and mutation rates among different PRRSV genotypes lead to mixed infections of multiple strains in my country, increasing the risk of missed detection and disease transmission. Therefore, a highly specific and sensitive kit capable of simultaneously detecting multiple pathogens is urgently needed. This invention develops a multiplex quantitative PCR detection kit for three types of porcine reproductive and respiratory syndrome viruses: PRRSV, NADC30, and NADC34. Based on sequences in GenBank, the N / Nsp2 gene sequences of PRRSV, NADC30, and NADC34 were cloned into the pUC57 vector, transformed into DH5α bacterial competent cells, and recombinant plasmids were extracted and sequenced for verification, serving as plasmid standards for multiplex quantitative PCR. The corresponding plasmid standards are named pUC57-PRRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34-Nsp2, respectively. OD values ​​were measured using a UV spectrophotometer. 260 The value is calculated using the formula: ([X(g / μL)DNA / DNA length (bp)×660]×6.02×10) 23 =Y(copies / μL)) converted to molar concentration, then diluted to 10 8 Copy / μL, store at -20℃, dilute before use.

[0036] II. Primer and Probe Design

[0037] Search and compare PRRSV N gene sequences (sequence numbers include: EU200966.1, EU200965.1, EU200964.1, EU200963.1, KY620012.1, KY620011.1, KY620010.1, KY620009.1, KY620007.1, KY620006.1, KY620005.1, KY620013.1) in GenBank; NADC30 Nsp2 gene sequences (MG847440.1, MG847427.1, MG847426.1); and NADC34 Nsp2 gene sequences (MN648057.1, MN648056.1). Multiple primer-probe combinations were designed for the conserved regions of these gene sequences, targeting each of the three targets. These combinations were then optimized and screened based on primer-probe specificity and amplification efficiency, ultimately selecting the optimal set of primers and probes. The PRRSV primer-probe design is shown in Table 1, with the 5' end labeled FAM and the 3' end labeled BHQ1. The NADC30 primer-probe design is shown in Table 2, with the 5' end labeled HEX and the 3' end labeled BHQ1. The NADC34 primer-probe design is shown in Table 3, with the 5' end labeled CY5 and the 3' end labeled BHQ3.

[0038] Table 1. PRRSV primers and probes

[0039]

[0040]

[0041] Table 2. NADC30 primers and probes

[0042]

[0043] Note: Y=T / C; R=A / G; M=A / C

[0044] Table 3. NADC34 primers and probes

[0045]

[0046] Note: R = A / G

[0047] III. Setting up internal standards

[0048] 18S rRNA is a conserved sequence widely found in various species, distributed across different tissues and cells, and is highly conserved and stable, making it an ideal internal standard. Clinically, 18S rRNA can be detected even in trace amounts of tissue samples. Phylogenetic trees were constructed by comparing the full-length 18S rRNA sequences of humans, chickens, gorillas, cattle, deer, horses, gibbons, ducks, mice, camels, sheep, guinea pigs, macaques, bats, rabbits, pigs, and whales from GenBank. The results showed that the 18S rRNA sequence identity among these species was 93.3%-99.9% (the compared sequences included: EU823286.1; FM165414.1; NR_046261.1; NR_046271.1; NR_036642.1; NR_146166.1; NR_145820.1; NR_04...). The sequences listed are: 6237.1; XR_004135034.1; XR_003587981.1; XR_004038418.1; XR_002788481.1; XR_002778881.1; XR_004246958.1; XR_004069086.1; XR_003493879.1; X00640.1). Therefore, it is shown that 18S rRNA is highly conserved in both mammals and birds, making it an ideal internal control gene. By comparing the above sequences to identify its conserved regions, primers were designed, and an amplification reaction was performed. Subsequently, the rRNA was cloned into the pUC57 vector and transformed into DH5α cells to obtain the recombinant clone pUC57-18S. We designed and screened a set of the best primers and probes, and based on the differences in a very small number of sequence sites, introduced a degenerate base into the probe and the downstream primer respectively (as shown in Table 4).

[0049] Table 4. Primers and probes for internal standard 18S rRNA

[0050]

[0051] Note: R = A / G

[0052] IV. Optimal reaction system and optimal reaction conditions

[0053] The reaction system and conditions for the designed primers and probes were optimized, including primer and probe reaction concentrations and Mn. 2+ Orthogonal experiments were conducted to determine the concentration and enzyme dosage. Primer concentrations were screened in the range of 0.2-0.8 μM, and probe concentrations were screened in the range of 0.1-0.4 μM, with increases of 0.1 μM for both. 2+ Screening was conducted with concentrations ranging from 0.5 to 5 mM, with each 0.5 mM increment representing a single step. Screening was conducted with enzyme dosages ranging from 1 to 10 U, with each 1 U increment representing a single step.

[0054] After conducting multiple rounds of orthogonal experiments, based on the differences and changes in Ct values ​​and fluorescence increments, a pair of highly specific and highly efficient primer-probe combination was selected, ultimately yielding an optimal primer-probe reaction mixture: Real-time PCR master mix (1×, Toyobo Biotechnology Co., Ltd.), Mn 2+ 2.5 mM, primer sequences 1, 2, 4, 5, 7, and 8 each 0.3 μM, primer sequences 10 and 11 each 0.2 μM, probe sequences 3, 6, and 9 each 0.2 μM, and probe sequence 12 0.15 μM.

[0055] The three-step reaction was compared with the two-step reaction (annealing, extension, and merging). The optimal annealing temperature was selected (temperature range 53℃-63℃, with a range of 2℃). The resulting reaction program with high specificity and high amplification efficiency was: 90℃ for 30s, 61℃ for 20min (reverse transcription step); 95℃ for 30s, 95℃ for 0s, 60℃ for 45s, for 40 cycles. Results were interpreted as follows: no Ct value indicates negative; Ct value <38 indicates positive; Ct value between 38-40 is questionable and requires retesting. If the retest Ct <40, a clear peak in the curve is observed, indicating a positive result; otherwise, it is negative.

[0056] Finally, the optimal reaction system and optimal reaction program were used to react with the pUC57-PRRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34-Nsp2 plasmid standards. The amplification effects were all good, and the results are as follows. Figure 1-3 As shown.

[0057] V. Specificity Test

[0058] Take inactivated equal volumes of porcine circovirus type 2 (PCV2), classical classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 3 (PCV3), extract viral RNA (or DNA) from the samples, add pathogen cDNA template, and detect the three different pathogens in the same reaction tube using the optimal reaction system and optimal reaction procedure described in (IV).

[0059] The results showed that the PRRSV / NADC30 / NADC34 multiplex quantitative PCR exhibited high specificity and no nonspecific reactions. Furthermore, the positive standards showed good reaction performance; the pUC57-PRRSV-N and pUC57-NADC30-Nsp2 plasmid standards both peaked at Ct value 22, while the pUC57-NADC34-Nsp2 plasmid standard peaked at Ct value 31. The internal standard also peaked at Ct value 33, indicating good quality control of the reaction (e.g., ...). Figure 4(As shown). Therefore, this kit will not produce false positives when used to detect porcine reproductive and respiratory syndrome (PRRS), ensuring the accuracy of the kit.

[0060] VI. Sensitivity Test

[0061] Using 10-fold serially diluted plasmid standards pUC57-PRRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34-Nsp2 as templates, corresponding primers and probes were added. Following the optimal reaction system and procedure described in section (IV), multiplex quantitative PCR was performed on three different pathogens (PRRSV, NADC30, and NADC34) in the same reaction tube. Results showed that the detection sensitivity for PRRSV, NADC30, and NADC34 was 10 copies of the plasmid standards (e.g., pUC57-PRRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34). Figure 5 (As shown). This demonstrates that the multiplex real-time PCR kit targeting PRRSV / NADC30 / NADC34 has high sensitivity and can detect trace amounts of virus in the early stages of infection. A search of recent relevant literature, as shown in the table below: Article 1 shows a detection limit of 4.6 × 10¹ copies / μL for HP-PRRSV2 and NL-PRRSV, and 4.6 × 10² copies / μL for PRRSV1 and CA-PRRSV2; Article 2 shows a detection limit of 24 copies for HP-PRRSV JXA1-R, HP-PRRSV, and C-PRRSV; Article 3 shows a detection limit of 1000 copies for CSFV / ASFV / PRRSV / PPV / PCV-2 / JEV. The sensitivity of these latest existing methods is significantly lower than that of this invention, indicating that this invention has significant advantages, especially in the simultaneous detection of three pathogens.

[0062] Table 5. Comparison of sensitivity of different research methods

[0063]

[0064]

[0065] VII. Repeatability Test

[0066] Intra-batch repeatability test: The plasmid standards pUC57-PRRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34-Nsp2 were serially diluted 10-fold, and 1×10⁻⁶ samples were taken from each. 6 ; 1×10 5 ; 1×10 3Three gradients, each with three replicates, were used to simultaneously perform multiplex quantitative PCR detection of three different pathogens (PRRSV, NADC30, and NADC34) in a single reaction tube, following the optimal reaction system and procedure described in section (IV). Results showed excellent intra-assay repeatability for all three gradients, with CV values ​​all less than 8% (of which 10...). 6 The intra-batch repeatability CV value of the plasmid was 2.1%; of which 10 5 The intra-batch repeatability CV value of the plasmid was 2.3%; of which 10 3 The intra-batch repeatability CV value of the plasmid was 3.7% (e.g., Figure 6 (As shown). Furthermore, the intra-batch repeatability is better than the relevant application standards, ensuring the stability of the reagent kit.

[0067] Inter-batch repeatability test: The plasmid standards pUC57-PPRSV-N, pUC57-NADC30-Nsp2, and pUC57-NADC34-Nsp2 were serially diluted 10-fold, and 1×10⁻⁶ samples were taken from each. 6 ; 1×10 5 ; 1×10 3 Three gradients were used for multiplex quantitative PCR of three different pathogens (PRRSV / NADC30 / NADC34) according to the optimal reaction system and procedure described in (IV). Two additional time points were selected for repeat experiments. The results showed that the batch-to-batch repeatability of all three gradients was excellent, with CV values ​​all less than 8% (of which 10...). 6 The batch-to-batch repeatability CV value of the plasmid was 4.1%; of which 10 5 The batch-to-batch repeatability CV value of the plasmid was 4.7%; of which 10 3 The batch-to-batch repeatability CV value of the plasmid was 5.7% (e.g., Figure 7 , 8 (As shown). Its batch repeatability is better than the relevant application standards, ensuring the stability of the reagent kit.

[0068] 8. Applications with MAOPA TM The technology's fully automated integrated response

[0069] The integrated structure used in this invention is as described in the applicant's patent application 202010147432.0 and Chinese patent application 201710429121.1, specifically as follows: The first chamber of the cartridge stores the sample lysis extraction solution (from top to bottom) for sample lysis and nucleic acid extraction; the second chamber of the cartridge stores sample washing solution I for the first nucleic acid washing; the third chamber of the cartridge stores sample washing solution II for the second nucleic acid washing; the primers and probes (sequences 1-9) for the target PRRSV / NADC30 / NADC34, the primers and probes (sequences 10-12) for the target internal standard, and the entire reaction premix are stored in the fourth chamber of the integrated detection reagent cartridge, and finally, the multiplex fluorescence quantitative PCR reaction is performed at the bottom of the cartridge.

[0070] This invention integrates nucleic acid extraction and molecular amplification reaction, truly realizing a fully enclosed automated reaction with sample input and result output. Manual operation only requires taking 600ul of sample and adding it to the integrated fully enclosed detection cartridge. The following reactions are carried out step by step in the integrated detection reagent cartridge: (1) Sample lysis, magnetic beads adsorb PRRSV / NADC30 / NADC34 genomic RNA, internal standard genes and species genes, etc.; (2) Magnetic beads carrying nucleic acid move directionally in the cartridge, gradually washing away non-nucleic acid samples such as proteins and impurities; (3) Magnetic beads carrying nucleic acid enter the amplification reaction area at the bottom of the cartridge, and perform multiplex fluorescent quantitative PCR under the action of reverse transcriptase. The 5' end of the PRRSV probe is labeled with FAM, the 5' end of the NADC30 probe is labeled with HEX, the 5' end of the NADC34 probe is labeled with CY5, and the 5' end of the internal standard probe is labeled with ROX. During the molecular amplification reaction, under the action of enzymes, the fluorescent labels at the 5' ends of various probes are hydrolyzed and released, and the instrument can capture and record the fluorescence signal. (4) The instrument analyzes the captured fluorescence signals and converts them into digital signals displayed on a visualization screen, thereby achieving intelligent output of real-time feedback of fluorescence curves. This enables real-time monitoring of the amplification of PRRSV / NADC30 / NADC34 pathogens and internal standards. To determine if the reaction is running normally, the internal standard is used as a reference. When the internal standard shows a typical logarithmic growth curve, it proves that all steps of the integrated system, including sample lysis, nucleic acid extraction, and quantitative fluorescence amplification, are normal. Its amplification curve is shown below. Figure 9 As shown.

[0071] Finally, it is understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. Primer pairs and probe combinations for simultaneous differential diagnosis of PRRSV, NADC30, and NADC34, including: (1) Primer pairs and probes for the PRRSV N gene, the primer pairs are shown in Sequence 1 and Sequence 2, and the probe sequence is shown in Sequence 3; (2) Primer pairs and probes for the NADC30 Nsp2 gene. The primer pairs are shown in Sequence 4 and Sequence 5, and the probe sequence is shown in Sequence 6. (3) Primer pairs and probes for the NADC34 Nsp2 gene, the primer pairs are shown in sequences 7 and 8, and the probe sequences are shown in sequence 9; The probe ends were modified with fluorescent labels: the 5' end of sequence 3 was modified with FAM and the 3' end with BHQ1; the 5' end of sequence 6 was modified with HEX and the 3' end with BHQ1; and the 5' end of sequence 9 was modified with CY5 and the 3' end with BHQ3. It also includes an internal standard primer-probe combination, which serves as an internal standard for identifying PRRSV, NADC30 and NADC34. The sequences of the internal standard primer pairs are shown in Sequence 10 and Sequence 11, respectively, and the probe sequence is shown in Sequence 12. The two ends of the internal standard probe were fluorescently labeled, with the 5' end modified with ROX and the 3' end modified with BHQ2.

2. A kit for simultaneously differentiating and diagnosing PRRSV, NADC30, and NADC34, characterized in that, The reagents in the kit include the primer pairs and probe combinations for the simultaneous differential diagnosis of PRRSV, NADC30 and NADC34 as described in claim 1.

3. The kit for simultaneous differential diagnosis of PRRSV, NADC30, and NADC34 as described in claim 2, characterized in that, Each primer pair and its corresponding probe are packaged individually or in combination.

4. The kit for simultaneous differential diagnosis of PRRSV, NADC30, and NADC34 as described in claim 3, characterized in that, The primer pairs and probes in the kit are embedded in a premixed solution, wherein the premixed solution includes Mn 2+ 2.5 mM; Primer and probe concentrations: 0.3 µM each for primers shown in sequence 1 and sequence 2, and 0.2 µM for probe shown in sequence 3; 0.3 µM each for primers shown in sequence 4 and sequence 5, and 0.2 µM for probe shown in sequence 6; 0.3 µM each for primers shown in sequence 7 and sequence 8, and 0.2 µM for probe shown in sequence 9; 0.2 µM each for primers shown in sequence 10 and sequence 11, and 0.15 µM for probe shown in sequence 12.

5. The kit for simultaneous differential diagnosis of PRRSV, NADC30, and NADC34 as described in claim 4, characterized in that, The reagents in the kit also include lysis extract and sample washing solution. The main components of the lysis extract are: 3 M Gu.HCl, 0.8 M NaCl, 30% isopropanol, 3% Tween 20, 1% Triton-X100, 0.3 mM EDTA, and 12 mM Tris.HCl.

6. The use of the reagents in the kit according to any one of claims 2 to 5 in the preparation of an integrated cartridge for the simultaneous identification and diagnosis of PRRSV, NADC30 and NADC34.

7. The application of the primer pair and probe combination for the simultaneous identification and diagnosis of PRRSV, NADC30 and NADC34 as described in claim 1 in the preparation of reagents for detecting PRRSV, NADC30 and NADC34.

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