Fluorescent PCR (polymerase chain reaction) detection kit for simultaneously identifying six PRRSV (porcine reproductive and respiratory syndrome virus) strains by single tube

Abstract

The invention relates to a kit for simultaneously detecting six PRRSV strains by a single tube based on a fluorescent quantitative PCR (Polymerase Chain Reaction) technology platform, and the kit can be used for compositely detecting the six PRRSV strains, including HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ and PRRSV-1. According to the invention, two technical principles of signal generation by the TaqMan hydrolysis probe and signal generation by hybridization of the specific fluorescent probe are combined, the same fluorescent channel can detect the fluorescent signal of the hydrolysis probe and also can detect a melting curve, and finally, the purpose of detecting two targets by the same fluorescent channel is achieved. The Ct value of an HP-PRRSV-like strain and the Tm value of a C-PRRSV strain are detected in an A488 channel, the Ct value of an NADC30-like strain and the Tm value of an internal standard are detected in an HEX channel, the Ct value of an NADC34-like strain and the Tm value of a QYYZ strain are detected in an ROX channel, and the Ct value of a PRRSV-1 strain is detected in a CY5 channel. The method has the advantages of high single-tube detection flux, high sensitivity, high detection speed, simplicity and convenience in operation and the like, and can provide powerful technical guarantee for monitoring, prevention and control of the PRRSV (Porcine Reproductive and Respiratory Syndrome Virus).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid detection, in particular to a fluorescent PCR detection kit for simultaneously identifying HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ and PRRSV-1 strains. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the most influential diseases in the world's pig industry, which is characterized by reproductive failure in pregnant sows and respiratory disease in piglets. PRRS pathogen is a single-stranded RNA virus, PRRSV (Porcine reproductive and respiratory syndrome virus, PRRSV), which is generally divided into PRRSV-1 type (European type) and PRRSV-2 type (American type). According to the genomic characteristics and pathogenicity of PRRSV, PRRSV-2 type prevalent in China can be further divided into five major strains: HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV and QYYZ. In recent years, the detection of PRRSV-1 type strains in China has shown an upward trend, and mixed infection with PRRSV-2 type strains often occurs, which has brought great difficulties to PRRS prevention and purification. At present, due to the limited cross-protection provided by commercial vaccines against different strains, the development of a differential diagnosis method for the major prevalent strains of PRRSV is of great significance for PRRS prevention and control.

[0003] Currently, the detection of PRRSV mainly relies on ELISA and conventional fluorescent quantitative PCR methods. The characteristics of these two methods are summarized as follows: (1) ELISA immunological analysis method is based on the specific binding of antigen and antibody. The biggest disadvantage is that it cannot be multiplexed, and the operation is complicated. Moreover, due to the sensitivity and specificity of the technology itself, false negatives or false positives may occur; (2) Conventional fluorescent quantitative PCR method can identify pathogens by detecting specific target DNA sequences, which has high sensitivity and good specificity. However, the mainstream instruments on the market currently support at most 4 channels of fluorescence, so the kit developed based on this platform can only achieve at most 4-fold detection even without adding internal standards, and cannot simultaneously achieve PRRSV diagnosis and typing.

[0004] This invention combines the signal generation principles of TaqMan hydrolysis probes and specific fluorescent probe hybridization. A single fluorescent channel can detect both the fluorescence signal of the hydrolysis probe and the melting curve, achieving the goal of detecting two targets with a single fluorescent channel. Based on this method, a single-tube fluorescent PCR detection kit was developed that can simultaneously identify HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ, and PRRSV-1 strains. This kit features high accuracy, low detection limit, good specificity, and ease of use. Furthermore, the inclusion of an internal standard and anti-contamination system effectively reduces false positives and false negatives. In conclusion, this invention provides a more accurate and efficient new method for monitoring porcine reproductive and respiratory syndrome (PRRS). Summary of the Invention

[0005] The purpose of this invention is to disclose a fluorescent PCR detection kit for simultaneously identifying six PRRSV strains in a single tube, in order to solve one or more technical problems existing in the existing methods and provide at least one beneficial option or create conditions.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention is to provide a fluorescent PCR detection kit.

[0007] The fluorescent PCR detection kit of the first aspect of the present invention includes a primer-probe mixture that simultaneously and specifically identifies six PRRSV strains, including HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ, and PRRSV-1.

[0008] In some embodiments of the present invention, the primer-probe mixture comprises: (1) a primer HP-F, a downstream primer HP-R, and a probe HP-P specifically amplifying HP-PRRSV-like primers, wherein the nucleotide sequence of HP-F is shown in SEQ ID No:1, the nucleotide sequence of HP-R is shown in SEQ ID No:2, and the nucleotide sequence of HP-P is shown in SEQ ID No:3; (2) a primer NADC30L-F, a downstream primer NADC30L-R, and a probe NADC30L-P specifically amplifying NADC30-like primers, wherein the nucleotide sequence of NADC30L-F is shown in SEQ ID No:4, the nucleotide sequence of NADC30L-R is shown in SEQ ID No:5, and the nucleotide sequence of NADC30L-P is shown in SEQ ID No:6; (3) a primer NADC34L-F, a downstream primer NADC34L-R, and a probe NADC34L-P specifically amplifying NADC34-like primers, wherein the nucleotide sequence of NADC34L-F is shown in SEQ ID No:6. As shown in No:7, the nucleotide sequence of NADC34L-R is shown in SEQ ID No:8, and the nucleotide sequence of NADC34L-P is shown in SEQ ID No:9; (4) Specific amplification of C-PRRSV upstream primer CPRRSV-F, downstream primer CPRRSV-R and probe CPRRSV-P, the nucleotide sequence of CPRRSV-F is shown in SEQ ID No:10, the nucleotide sequence of CPRRSV-R is shown in SEQ ID No:11, and the nucleotide sequence of CPRRSV-P is shown in SEQ ID No:12; (5) Specific amplification of QYYZ upstream primer QYYZ-F, downstream primer QYYZ-R and probe QYYZ-P, the nucleotide sequence of QYYZ-F is shown in SEQ ID No:13, the nucleotide sequence of QYYZ-R is shown in SEQ ID No:14, and the nucleotide sequence of QYYZ-P is shown in SEQ ID No:9. As shown in No:15; (6) Specific amplification of PRRSV-1 using upstream primer EU-F, downstream primer EU-R, and probe EU-P, wherein the nucleotide sequence of EU-F ​​is shown in SEQ ID No:16, the nucleotide sequence of EU-R is shown in SEQ ID No:17, and the nucleotide sequence of EU-P is shown in SEQ ID No:18. Through the above 6 primer pairs and probes, the fluorescent PCR detection kit can achieve the typing amplification and specific detection of 6 different PRRSV strains in a single multiplex amplification system.

[0009] In some embodiments of the present invention, the primer-probe mixture further includes an upstream primer IC-F, a downstream primer IC-R, and a probe IC-P for specifically amplifying the internal control gene. The nucleotide sequence of IC-F is shown in SEQ ID No:19, the nucleotide sequence of IC-R is shown in SEQ ID No:20, and the nucleotide sequence of IC-P is shown in SEQ ID No:21.

[0010] In some embodiments of the present invention, the 5' end of each probe is modified with a fluorescent group, and the 3' end is modified with a minor groove binder or a non-fluorescent quencher. A minor groove binder (MGB) is a chemical group or molecule capable of selectively binding non-covalently to the minor groove of a DNA molecule. After binding to the DNA minor groove, MGB stabilizes the DNA double-stranded structure primarily through van der Waals forces and hydrophobic interactions. Modification with MGB can shorten the probe length, improve the specificity and stability of probe binding to the target sequence, and the use of a non-fluorescent quencher can greatly eliminate background fluorescence generated by traditional quenchers, improving the signal-to-noise ratio. Non-fluorescent quenchers have the advantages of no background interference, high quenching efficiency, and strong stability.

[0011] In some embodiments of the present invention, the 5' end of the HP-P is modified with A488, and the 3' end is modified with MGB; the 5' end of the NADC30L-P is modified with TET, and the 3' end is modified with MGB; the 5' end of the NADC34L-P is modified with ROX, and the 3' end is modified with MGB; the 5' end of the CPRRSV-P is modified with A488, and the 3' end is modified with BHQ1; the 5' end of the QYYZ-P is modified with ROX, and the 3' end is modified with BHQ2; the 5' end of the EU-P is modified with CY5, and the 3' end is modified with BHQ2.

[0012] Furthermore, the final concentrations of each primer and probe in the primer-probe mixture in the reaction system are as follows: HP-F and HP-R are both 0.4 μmol / L; HP-P is 0.20 μmol / L; NADC30L-F and NADC30L-R are both 0.6 μmol / L; NADC30L-P is 0.15 μmol / L; NADC34L-F and NADC34L-R are both 0.5 μmol / L; NADC34L-P is 0.2 μmol / L; CPRRSV-F is 0.04 μmol / L, CPRRSV-R is 0.4 μmol / L, and CPRRSV-P is 0.2 μmol / L; QYYZ-F is 0.06 μmol / L, and QYYZ-R is 0.6 μmol / L. The final concentrations of QYYZ-P and IC-P are as follows: QYYZ-P: 0.3 μmol / L; EU-F ​​and EU-R: 0.4 μmol / L; EU-P: 0.12 μmol / L; IC-F: 0.04 μmol / L; IC-R: 0.3 μmol / L; and IC-P: 0.2 μmol / L.

[0013] In some embodiments of the present invention, the fluorescent PCR detection kit further includes a premixed PCR MIX, which comprises the following components: hot-start Taq enzyme, heat-sensitive UDG enzyme, reverse transcriptase, Tris-HCl, ammonium sulfate, magnesium chloride, dATPs, dGTPs, dCTPs, dUTPs, prololan polysaccharide, and bovine serum albumin. The prololan polysaccharide provides a more stable and favorable microenvironment for other key components in the PCR reaction system (such as DNA polymerase), reducing the damage to these active molecules caused by high temperatures, especially when the reaction system is small and easily evaporates.

[0014] Furthermore, the concentrations of each component in the premixed PCR MIX are as follows: hot-start Taq enzyme concentration is 0.2 U / μL, heat-sensitive UDG enzyme concentration is 0.05 U / μL, reverse transcriptase concentration is 0.2 U / μL, Tris-HCl at pH 8.1 concentration is 10 mmol / L, ammonium sulfate concentration is 10 mmol / L, magnesium chloride concentration is 5 mmol / L, dATPs concentration is 0.2 mmol / L, dGTPs concentration is 0.2 mmol / L, dCTPs concentration is 0.2 mmol / L, dUTPs concentration is 0.5 mmol / L, prolan polysaccharide concentration is 50 mmol / L, and bovine serum albumin concentration is 0.5 μg / μL.

[0015] In some embodiments of the present invention, the fluorescent PCR detection kit further includes a negative control and a positive control, wherein the negative control is sterile deionized water and the positive control is a mixture of recombinant plasmids of HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ and PRRSV-1.

[0016] The second aspect of the present invention is to provide the application of the fluorescent PCR detection kit described in the first aspect of the present invention in PRRSV detection or PRRSV typing for purposes other than disease diagnosis or treatment. Attached Figure Description

[0017] Figure 1 This is the detection chromatogram of the positive control sample in Example 5; Figure 2 This is the detection spectrum of the negative control sample in Example 5; Figure 3 This is the detection spectrum of the C-PRRSV strain before the probe sequence was modified in the comparative example; Figure 4 This is the detection pattern after the probe sequence of the C-PRRSV strain was modified in the comparison. Detailed Implementation

[0018] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.

[0019] Example 1: Primer and probe design for six PRRSV strains and internal standards The whole genome sequences of HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ, and PRRSV-1 strains were downloaded from the GeneBank database. Multiple sequence alignment was used to align conserved gene sequences within the strains and screen for specific gene sequences between strains. The GeneBank sequence numbers extracted from the six strains were FJ548853.1, MH651746.1, MN648450.1, EF536003.1, JQ308798.1, and MK024327.1, respectively. The pig housekeeping gene HPRT1 (NM_001032376.2) was selected as the template sequence for the internal standard. Based on the above DNA templates, primers and probes for six PRRSV strains and internal standards were designed using Oligo 7.0 software. The performance parameters of individual target primers and probes were evaluated using Primer 6.0 software. Finally, the dimer and hairpin structure between the composite primers and probes were analyzed using MF Primer software. Since the technical principle used in this invention is different from the conventional real-time PCR method, the principles of primer and probe design, especially the design of target probes using melting curve analysis, are very different from those of conventional PCR. The specific design principles are as follows: (1) The probe design length should be between 15 and 30 bp to ensure the binding stability between the probe and the template; (2) The position of the probe on the DNA sequence should not be close to the same-direction primer; (3) The Tm value of the probe should be lower than the Tm value of the primer; (4) It is best to design a 4-5 bp complementary sequence at the 5' and 3' ends of the probe to reduce the background signal of the reaction system. In addition to the differences in single target probe design, the final evaluation of multiple primers and probes is also the key to PCR success. It is necessary to ensure that individual targets have similar reaction efficiencies, that primers and probes do not form dimers (especially complementary pairings at the 3' end exceeding 3 bp), and that each primer and probe has similar Tm values ​​and base composition.

[0020] The primer and probe design of this invention cannot follow conventional real-time PCR design methods. Only innovative thinking combined with extensive experiments can successfully summarize the design rules. Meanwhile, as the number of detection targets in the multiplex amplification system increases, evaluating the interference between multiple primers and probes also becomes a significant challenge, requiring numerous experiments to screen for suitable primer and probe combinations. The primer and probe sequences for six PRRSV strains and internal standards are shown in Table 1.

[0021] Table 1 - Primer and probe sequences for six PRRSV strains and internal standards

[0022] Example 2: Primer and probe concentration adjustment This invention allows for the detection of six PRRSV strains in a single tube, and the same fluorescent channel can detect two signals: a hydrolysis probe signal and a melting curve signal. Conventional methods use non-specific fluorescent dyes to create melting curves, which have poor specificity and are difficult to implement for multiplex detection. This invention innovatively uses specific fluorescent probes to hybridize with amplification products to generate melting curve signals, achieving not only high specificity but also dual detection in a single-color channel. However, adjusting primer and probe concentrations in this method is challenging. First, as the amount of target molecules in the quantitative PCR system increases, competition between primers and probes makes controlling the amplification efficiency of each target more difficult, requiring numerous repeated experiments to adjust the concentrations of primers and probes for each target. Second, the fusion of two technical principles requires balancing the detection effects of both the hydrolysis probe and the melting curve, also necessitating numerous repeated experiments to optimize primer and probe concentrations. The primer and probe concentrations for the six PRRSV strains and the internal standard are shown in Table 2.

[0023] Table 2 - Primer and probe concentrations for six PRRSV strains and internal standards

[0024] Example 3: PCR reaction MIX optimization The polymerase, reverse transcriptase, UDG enzyme, buffer concentration and pH, MgCl2 to dNTP ratio, and type and concentration of enhancer in the PCR reaction mix all affect the PCR amplification results. DNA polymerase catalyzes the PCR reaction; too low a final concentration inhibits inefficient target amplification, while too high a concentration leads to nonspecific amplification. Reverse transcriptase is used to reverse transcribe viral RNA into DNA; its concentration needs to be carefully controlled to improve reverse transcription efficiency without affecting PCR amplification efficiency. UDG enzyme is a key ingredient in preventing PCR amplification product contamination; suitable heat-sensitive UDG enzymes must be selected to prevent target product degradation during amplification, and enzyme concentration must be controlled to reduce its inhibitory effect on PCR amplification. To enhance the stability and sensitivity of PCR amplification, appropriate MgCl2, dNTP concentrations, and enhancers need to be added to the PCR reaction mix. Too high a MgCl2 concentration leads to nonspecific amplification, while too low a concentration reduces PCR amplification efficiency; the MgCl2 to dNTP ratio is also a key factor affecting PCR amplification efficiency. Different PCR enhancers have their own characteristics, and only at their suitable concentrations can they enhance PCR amplification. Too low a concentration will have little effect, while too high a concentration will inhibit PCR amplification. Therefore, the components in various PCR reaction mixes need to undergo extensive experimental screening and concentration optimization to effectively enhance PCR amplification. The components and concentrations of PCR reaction mixes are shown in Table 3.

[0025] Table 3 - PCR reaction MIX components and concentrations

[0026] Example 4: Program Optimization PCR contamination is one of the most common problems in molecular laboratories, with PCR product contamination being the most serious. To reduce the probability of PCR product contamination, the kit of this invention introduces an UDG enzyme anti-contamination system, which also includes an UDG enzyme digestion program before the PCR amplification reaction. Because the kit of this invention uses a one-step reverse transcription system, the reverse transcriptase reaction time is increased after the UDG enzyme digestion reaction, and the reaction time is optimized to balance reverse transcription efficiency and reaction time. To further shorten the reaction time, this invention uses a rapid PCR amplification program, combining the annealing and extension steps into one, changing the three-step program to a two-step program, and reducing the reaction time of each step. Annealing temperature is a key factor affecting PCR amplification efficiency and specificity. Appropriately lowering the annealing temperature can improve amplification efficiency, but non-specific amplification, especially primer dimers, will increase. Conversely, raising the annealing temperature can improve specificity, but may cause a decrease in PCR amplification efficiency. To take all these factors into account, this invention has systematically debugged and optimized the anti-contamination program, reverse transcription program, and in-cycle amplification program. The PCR amplification program is shown in Table 4 below: Table 4 - PCR Amplification Procedure

[0027] Example 5: Positive and negative control sample testing This invention's kit can detect both the fluorescence signal of TaqMan probes and melting curves in the same fluorescence channel. Specifically, the A488 channel can simultaneously detect the Ct value of HP-PRRSV-like strains and the Tm value of C-PRRSV strains; the HEX channel can simultaneously detect the Ct value of NADC30-like strains and the Tm value of the internal standard; the ROX channel can simultaneously detect the Ct value of NADC34-like strains and the Tm value of QYYZ strains; and the CY5 channel can detect the Ct value of PRRSV-1 strains, ultimately achieving single-tube detection of seven targets. The detection chromatogram of the positive control sample is shown below. Figure 1 As shown, the detection chromatogram of the negative control sample is as follows: Figure 2 As shown.

[0028] Example 6: Sensitivity assessment and determination of positive cutoff value The concentration of the six positive control samples described in this invention is 10. 6 Copy / mL. The positive control sample was diluted to 10 copies / mL using a 10-fold serial dilution method. 3 10 2 and 10 1Copy / mL. The lowest concentration of the quality control sample capable of detecting all six strains is 10. 2 The limit of detection (LOD) was set at copies / mL. The Ct value at this limit was used as the positive cutoff value for HP-PRRSV-like, NADC30-like, NADC34-like, and PRRSV-1 strains, respectively. The Tm range was used as the positive cutoff value for C-PRRSV strains, QYYZ strains, and the internal standard IC. The positive cutoff values ​​are shown in Table 5.

[0029] Table 5 - Positive Cutoff Values

[0030] Comparative example: C-PRRSV strain probe sequence modification During the primer and probe testing process of this invention, in addition to the melting curve, the C-PRRSV strain also exhibits an amplification curve and Ct value for the hydrolysis probe. This ultimately interferes with the detection of HP-PRRSV-like strains in the A488 channel, leading to false positives for HP-PRRSV-like strains during the detection process. To eliminate the amplification signal of the C-PRRSV strain hydrolysis probe in the results, through numerous repeated experiments, it was found that adding a complementary fragment to the 3' end of the C-PRRSV strain probe sequence at the 5' end can solve the above problem. The detection pattern of the C-PRRSV strain probe sequence before modification is shown below. Figure 3 As shown, the detection pattern after modifying the C-PRRSV strain probe sequence is as follows: Figure 4 As shown in Table 6, the sequences of the C-PRRSV strain probe before and after modification are shown in Table 6.

[0031] Table 6 - Sequences of C-PRRSV strain probes before and after modification

Claims

1. A fluorescent PCR detection kit, characterized in that, The mixture includes a primer-probe mixture that simultaneously and specifically identifies six PRRSV strains, including HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ, and PRRSV-1.

2. The fluorescent PCR detection kit according to claim 1, characterized in that, The primer-probe mixture comprises: The upstream primer HP-F, the downstream primer HP-R, and the probe HP-P are specifically amplified to resemble HP-PRRSV. The nucleotide sequence of HP-F is shown in SEQ ID No:1, the nucleotide sequence of HP-R is shown in SEQ ID No:2, and the nucleotide sequence of HP-P is shown in SEQ ID No:

3. The upstream primer NADC30L-F, the downstream primer NADC30L-R, and the probe NADC30L-P specifically amplify NADC30-like molecules. The nucleotide sequence of NADC30L-F is shown in SEQ ID No:4, the nucleotide sequence of NADC30L-R is shown in SEQ ID No:5, and the nucleotide sequence of NADC30L-P is shown in SEQ ID No:

6. The upstream primer NADC34L-F, the downstream primer NADC34L-R, and the probe NADC34L-P are specifically amplified to resemble NADC34. The nucleotide sequence of NADC34L-F is shown in SEQ ID No:7, the nucleotide sequence of NADC34L-R is shown in SEQ ID No:8, and the nucleotide sequence of NADC34L-P is shown in SEQ ID No:

9. The upstream primer CPRRSV-F, the downstream primer CPRRSV-R, and the probe CPRRSV-P are used to specifically amplify C-PRRSV. The nucleotide sequence of CPRRSV-F is shown in SEQ ID No:10, the nucleotide sequence of CPRRSV-R is shown in SEQ ID No:11, and the nucleotide sequence of CPRRSV-P is shown in SEQ ID No:

12. The upstream primer QYYZ-F, the downstream primer QYYZ-R, and the probe QYYZ-P are specifically amplified by QYYZ. The nucleotide sequence of QYYZ-F is shown in SEQ ID No:13, the nucleotide sequence of QYYZ-R is shown in SEQ ID No:14, and the nucleotide sequence of QYYZ-P is shown in SEQ ID No:

15. The upstream primer EU-F, the downstream primer EU-R, and the probe EU-P are used to specifically amplify PRRSV-1. The nucleotide sequence of EU-F ​​is shown in SEQ ID No:16, the nucleotide sequence of EU-R is shown in SEQ ID No:17, and the nucleotide sequence of EU-P is shown in SEQ ID No:

18.

3. The fluorescent PCR detection kit according to claim 2, characterized in that, The primer-probe mixture further includes an upstream primer IC-F, a downstream primer IC-R, and a probe IC-P for specifically amplifying the internal control gene. The nucleotide sequence of IC-F is shown in SEQ ID No:19, the nucleotide sequence of IC-R is shown in SEQ ID No:20, and the nucleotide sequence of IC-P is shown in SEQ ID No:

21.

4. The fluorescent PCR detection kit according to claim 2 or 3, characterized in that, Each probe has a fluorescent group modified at its 5' end and a minor groove conjugate or a non-fluorescent quencher modified at its 3' end.

5. The fluorescent PCR detection kit according to claim 4, characterized in that, The HP-P has its 5' end modified with A488 and its 3' end modified with MGB; the NADC30L-P has its 5' end modified with TET and its 3' end modified with MGB; the NADC34L-P has its 5' end modified with ROX and its 3' end modified with MGB; the CPRRSV-P has its 5' end modified with A488 and its 3' end modified with BHQ1; the QYYZ-P has its 5' end modified with ROX and its 3' end modified with BHQ2; and the EU-P has its 5' end modified with CY5 and its 3' end modified with BHQ2.

6. The fluorescent PCR detection kit according to claim 2, characterized in that, The final concentrations of each primer and probe in the primer-probe mixture in the reaction system are as follows: HP-F and HP-R are both 0.4 μmol / L; HP-P is 0.20 μmol / L; NADC30L-F and NADC30L-R are both 0.6 μmol / L; NADC30L-P is 0.15 μmol / L; NADC34L-F and NADC34L-R are both 0.5 μmol / L; NADC34L-P is 0.2 μmol / L; CPRRSV-F is 0.04 μmol / L, CPRRSV-R is 0.4 μmol / L, and CPRRSV-P is 0.2 μmol / L; QYYZ-F is 0.06 μmol / L, and QYYZ-R is 0.6 μmol / L. The final concentration of QYYZ-P is 0.3 μmol / L; the final concentrations of EU-F ​​and EU-R are 0.4 μmol / L; and the final concentration of EU-P is 0.12 μmol / L.

7. The fluorescent PCR detection kit according to claim 1, characterized in that, It also includes a premixed PCR MIX, which comprises the following components: hot-start Taq enzyme, heat-sensitive UDG enzyme, reverse transcriptase, Tris-HCl, ammonium sulfate, magnesium chloride, dATPs, dGTPs, dCTPs, dUTPs, prololan polysaccharide, and bovine serum albumin.

8. The fluorescent PCR detection kit according to claim 7, characterized in that, The concentrations of each component in the premixed PCR MIX are as follows: hot-start Taq enzyme concentration is 0.2 U / μL, heat-sensitive UDG enzyme concentration is 0.05 U / μL, reverse transcriptase concentration is 0.2 U / μL, Tris-HCl at pH 8.1 concentration is 10 mmol / L, ammonium sulfate concentration is 10 mmol / L, magnesium chloride concentration is 5 mmol / L, dATPs concentration is 0.2 mmol / L, dGTPs concentration is 0.2 mmol / L, dCTPs concentration is 0.2 mmol / L, dUTPs concentration is 0.5 mmol / L, prolan polysaccharide concentration is 50 mmol / L, and bovine serum albumin concentration is 0.5 μg / μL.

9. The fluorescent PCR detection kit according to claim 1, characterized in that, It also includes negative and positive control samples, wherein the negative control sample is sterile deionized water and the positive control sample is a mixture of recombinant plasmids of HP-PRRSV-like, NADC30-like, NADC34-like, C-PRRSV, QYYZ and PRRSV-1.

10. The use of the fluorescent PCR detection kit according to any one of claims 1 to 9 in PRRSV detection or PRRSV typing for purposes other than disease diagnosis or treatment.

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