CN105779650A - Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains - Google Patents

Abstract

The invention discloses a triple fluorescent quantitative PCR primer, probe and kit for distinguishing classic pseudorabies virus strains, mutant strains and Bartha-K61 vaccine strains. The present invention firstly discloses primers and probes for triple fluorescence quantitative PCR for distinguishing classic strains of pseudorabies virus, mutant strains and Bartha-K61 vaccine strains, wherein the primer sequences are SEQ ID No.8-9, SEQ ID No.14 -15, the probe sequence is shown in SEQ ID No.1, 4 and 6. The present invention further discloses a triple fluorescent quantitative PCR detection kit, which is based on three TaqMan probes labeled with different fluorescent signals, can accurately identify pseudorabies virus classic strains, mutant strains and Bartha-K61 vaccine strains, and has good The sensitivity and specificity can be applied to the differential detection of pseudorabies virus strains.

Description

Primers, probes and kits for triplex quantitative PCR for identification of pseudorabies virus strains

technical field

The present invention relates to primers and probes for triple fluorescent quantitative PCR for distinguishing classic strains of pseudorabies virus, mutant strains and Bartha-K61 vaccine strains, and also relates to a method for distinguishing classic strains of pseudorabies virus, mutant strains and Bartha-K61 vaccine strains The triple fluorescent quantitative PCR detection kit belongs to the field of detection and identification of different strains of pseudorabies virus.

Background technique

Pseudorabies (PR), also known as Ojecki's disease, is an acute infectious disease of livestock and wild animals caused by pseudorabies virus (PRV) infection, and is one of the pathogens that cause major economic losses in the world's pig industry . Pigs are the storage host and main source of infection of the disease, and the clinical symptoms are mainly characterized by fever, nervous system and respiratory disturbance. Lactating piglets and young pigs are the most sensitive to the virus. After infection, body weight and appetite decrease, and the mortality rate is 50-100%. PRV, also known as porcine herpesvirus type 1, belongs to the family Herpesviridae. PRV is a double-stranded DNA virus with a length of about 143kb and can encode 70-100 proteins (MettenleiterTC. 2000. Aujeszky's disease (Pseudorabies) virus: the virus and molecular pathogenesis-state of heart. VetRes31:99-115.).

Since the end of 2011, new outbreaks of pseudorabies have successively broken out in pig farms in several provinces in China. Even if they have been immunized with the Bartha-K61 attenuated vaccine, they still cannot effectively resist the new outbreaks. Some studies have shown that the outbreak was caused by a PRV variant. Compared with the classic PRV strains, the mutant strains had regular insertions and deletions in multiple genes, such as gE, gI and gB genes.这些变异株在进化上形成了一个新的分支(AnTQ,etal.,2013.PseudorabiesvirusvariantinBartha-K61-vaccinatedpigs,China,2012.EmergInfectDis19:1749–1755；LuoY,etal.,2014.PathogenicityandgenomiccharacterizationofapseudorabiesvirusvariantisolatedfromBartha-K61-vaccinatedswinepopulationinChina.VetMicrobiol174 :107–115.). At present, the Bartha-K61 vaccine strain can no longer provide sufficient protection against PRV mutant strains, so PRV mutant strains pose a serious threat to China's pig industry.

The classic PRV detection method is virus isolation and immunofluorescence analysis (PensaertMB, KlugeJP.1989. . Immunoperoxidase study of Aujeszky's disease virus in pigs. ResVetSci32:294–302.), but these two methods have a long experimental cycle and high experimental consumption. At present, a variety of PRV detection methods have been established, such as polymerase chain amplification reaction (PRV), loop-mediated isothermal amplification reaction (Loop-mediated isothermal amplification, LAMP), electrochemical reaction based on colloidal gold, immunochromatographic test paper, enzyme Linked immunosorbent assay (ELISA) and fluorescent quantitative PCR (ZanellaEL, et al., 2012. Evaluation of real-time polymerase chain reaction assay for Pseudorabies virus surveillance purposes. JVet Diagn Invest24:739-745.). However, these methods cannot discriminate between classic and variant strains of PRV. Although virus isolation combined with DNA sequencing can be used to identify and distinguish different strains, this method is time-consuming and laborious.

Therefore, the establishment of a new triple fluorescent quantitative PCR detection method that can quickly distinguish and detect PRV classic strains, mutant strains and Bartha-K61 vaccine strains will be of great significance for the differential detection of PRV strains and the response to the increasingly serious PR epidemic. and application value.

Contents of the invention

The technical problem to be solved by the present invention is to provide a kind of primer and probe for the triple fluorescent quantitative PCR for distinguishing pseudorabies virus classic strain, mutant strain and Bartha-K61 vaccine strain;

Another technical problem to be solved by the present invention is to provide a triple fluorescence quantitative PCR detection kit for distinguishing classic strains, mutant strains and Bartha-K61 vaccine strains of pseudorabies virus.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

In the present invention, by analyzing the gene sequences of different strains of pseudorabies virus (PRV), multiple sets of common primer pairs for PRV classic strains and mutant strains are designed at the gene deletion site of the Bartha-K61 vaccine strain (Bartha-K61 vaccine strain is a gE/gI deletion strain) and probes, multiple sets of primer pairs and probes were designed on the sequence of the Bartha-K61 vaccine strain. Concretely, the primers for triple fluorescent quantitative PCR used to identify pseudorabies virus classic strain, mutant strain and Bartha-K61 vaccine strain in the present invention include: a primer pair 1 composed of the nucleotide sequences shown in SEQIDNo.8 and SEQIDNo.9 , any pair of primers 3 formed of the nucleotide sequences shown in SEQIDNo.10 and SEQIDNo.11, the primer pair 2, the nucleotide sequence shown in SEQIDNo.12 and SEQIDNo.13 (the above 3 pairs of primers A pair of common primers for classic strains and mutant strains), and a pair of primers 4 consisting of nucleotide sequences shown in SEQIDNo.14 and SEQIDNo.15 (a pair of primers for detecting the Bartha-K61 vaccine strain).

The probes include: any one of the nucleotide sequences shown in SEQIDNo.1, SEQIDNo.2 or SEQIDNo.3 (the above three probes are used to detect the classic strain of pseudorabies virus), SEQIDNo.4 or SEQIDNo. Any one of the nucleotide sequences shown in 5 (the above two probes are used to detect pseudorabies virus variants), and any one of the nucleotide sequences shown in SEQIDNo.6 or SEQIDNo.7 (the above two A probe was used to detect the Bartha-K61 vaccine strain). Wherein, the nucleotide sequence of the probe shown in SEQIDNo.1, SEQIDNo.2 or SEQIDNo.3 is marked with a FAM fluorescent reporter group at the 5' end, and a BHQ1 fluorescence quencher group at the 3' end; The nucleotide sequence of the probe shown in SEQIDNo.4 or SEQIDNo.5 is marked with HEX fluorescent reporter group at the 5' end and BHQ1 fluorescent quenching group at the 3' end respectively; the nucleotide sequence is SEQIDNo.6 Or the 5' end of the probe shown in SEQ ID No.7 is marked with Cy5 fluorescent reporter group, and the 3' end is marked with BHQ3 fluorescence quencher group.

The present invention optimizes the different combinations of the above-mentioned primers and probes. The results show that the probes of the nucleotide sequences shown in SEQIDNo.1, SEQIDNo.4 and SEQIDNo.6 are used, as well as the probes shown in SEQIDNo.8 and SEQIDNo.9 The primer pair 1, the primer pair 4 composed of the nucleotide sequence shown in SEQIDNo.14 and SEQIDNo.15 can be used to treat three kinds of pseudorabies virus classic strain, mutant strain and Bartha-K61 vaccine strain. The virus strains can be detected and differentiated at the same time, and the CT value is the lowest, the detection limit is the lowest, and the sensitivity is the highest; while other combinations of primers and probes cannot detect and distinguish the classic strain, variant strain and Bartha-K61 vaccine strain at the same time.

Through the design and optimization of the primers and probes of the present invention, it can be seen that because the genome sequences of different strains of the same virus are extremely similar, especially the amino acid structures of the classic PRV strain and the mutant strain are basically the same, and the main genes only have individual amino acid insertions and deletions. The origin is extremely high, so it is difficult to design probes for simultaneously identifying three or more different strains of PRV. The present invention strategically designs two probes (shown in SEQIDNo.1 and SEQIDNo.4) at the gene deletion site of the Bartha-K61 vaccine strain to distinguish classic strains and mutant strains; at the same time, the second probe is designed at the connection position of the Bartha-K61 deletion site Three probes (shown in SEQIDNo.6) are used to detect vaccine strains. The present invention uses the above three probes to establish a triple fluorescent quantitative PCR method to identify and detect PRV classic strains, mutant strains and Bartha-K61 vaccine strains.

Based on three kinds of TaqMan probes (FAM (specific to classic strains), HEX (specific to mutant strains) and Cy5 (specific to vaccine strains)) with different fluorescent signals labeled, the triple fluorescent quantitative PCR method of the present invention can distinguish PRV classic strains simultaneously (SC strain), mutant strain (TJ strain) and Bartha-K61 vaccine strain. The method has strong specificity, and no fluorescent signal was detected for CSFV, PCV2, PPV and PRRSV. The detection limits of the triple fluorescent quantitative PCR method of the present invention for SC strain, TJ strain and Bartha-K61 are 0.5TCID ₅₀ , 0.2TCID ₅₀ and 0.05TCID ₅₀ , respectively. Repeatability tests within and between groups showed that the coefficient of variation of the method was less than 3%.

The triple fluorescent quantitative PCR method of the present invention can accurately identify different strains of PRV, and the identification result is the same as the sequencing result. At the same time, in the case of PRVSC strain, TJ strain and Bartha-K61 vaccine strain mixed in pairs or in threes, the method can also accurately identify the strain type in the virus mixture. The triple fluorescence quantitative PCR method and the virus isolation method of the present invention have detection coincidence rates of 100%, 99.5% and 100% for classic strains, variant strains and vaccine strains respectively. In addition, the viral loads of different PRVs in various organs of pigs can also be detected by this method.

The classic strains of pseudorabies virus in the present invention include but not limited to: SC strain or Min-A strain of pseudorabies virus; the variant strains include but not limited to: TJ strain of pseudorabies virus, HLJMDJ2013 strain, HeBLP2014 strain or BJKJZ2015 strain.

The primers and probes of the present invention can be applied to prepare reagents or materials for distinguishing classic strains, variant strains and Bartha-K61 vaccine strains of pseudorabies virus.

The present invention further discloses a triple fluorescent quantitative PCR detection kit for distinguishing pseudorabies virus classic strain, mutant strain and Bartha-K61 vaccine strain, comprising: primers, probes, PremixExTaqProbeqPCR and double distilled water; wherein said primers are composed of SEQIDNo The primer pair 1 that the nucleotide sequence shown in .8 and SEQIDNo.9 forms, the primer pair 4 that the nucleotide sequence shown in SEQIDNo.14 and SEQIDNo.15 forms; Described probe is SEQIDNo.1, SEQIDNo. .4 and the nucleotide sequence shown in SEQ ID No.6. Wherein, the nucleotide sequence is that the 5' end of the probe shown in SEQIDNo.1 is marked with a FAM fluorescent reporter group, and the 3' end is marked with a BHQ1 fluorescence quenching group; the nucleotide sequence is shown in SEQIDNo.4 The 5' end of the probe is marked with a HEX fluorescent reporter group, and the 3' end is marked with a BHQ1 fluorescent quencher group; the 5' end of the probe whose nucleotide sequence is shown in SEQIDNo.6 is marked with a Cy5 fluorescent reporter group , the 3' end is labeled with a BHQ3 fluorescence quencher.

The triple fluorescent quantitative PCR detection kit of the present invention also includes dimethyl sulfoxide (DMSO). Wherein, the amount of dimethyl sulfoxide is 0%-10% of the total volume of the triple fluorescent quantitative PCR reaction system composed of primers, probes, PremixExTaqProbeqPCR, double distilled water, genomic DNA to be tested and dimethyl sulfoxide , preferably 2.5%-10%, most preferably 5%.

It has been reported in the literature that adding DMSO to the common PCR reaction system can increase the sensitivity of the reaction. However, fluorescent quantitative PCR itself is a highly sensitive detection method, and the addition of DMSO may affect the reading of fluorescent signals. Pseudorabies virus double-stranded DNA binds relatively tightly. When denatured at 95°C, the double-stranded DNA cannot be completely opened, and primers and probes cannot be combined with double-stranded DNA and amplified, which reduces the recognition and identification ability of the probe-primer pair reaction. In the present invention, DMSO is added to the triple fluorescent quantitative PCR reaction system, which enhances the identification ability of the probe and significantly improves the sensitivity of the method; when the added amount of DMSO is 5% (v/v), the CT value is the lowest and the sensitivity The highest, the accuracy rate of discrimination reaches 100%.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

The present invention strategically designs two probes at the gene deletion site of the Bartha-K61 vaccine strain to distinguish classic strains and mutant strains, and at the same time designs a third probe at the connection position of the Bartha-K61 deletion site for detection of vaccine strains . The invention utilizes the designed three probes to establish a triple fluorescent quantitative PCR method for distinguishing classic pseudorabies virus strains, mutant strains and Bartha-K61 vaccine strains. In addition, the present invention adds DMSO to the triple fluorescent quantitative PCR reaction system, which greatly improves the sensitivity of the method. Based on three TaqMan probes labeled with different fluorescent signals, the triple fluorescent quantitative PCR method of the present invention can identify PRV classic strains, mutant strains and Bartha-K61 vaccine strains, has good sensitivity and specificity, and is time-consuming and low-cost , with high accuracy, can be applied to the differential detection of PRV strains and to deal with the increasingly serious PR epidemic.

Description of drawings

Figure 1 is the selection of triple fluorescent quantitative PCR primers and probes; A-F are the amplification curves of scheme 1-scheme 6 respectively;

Fig. 2 is the specific primers and probes for amplifying PRV classic strains and variant strains; wherein, the design positions of primers and probes for PRV classic strains and variant strains are in the dashed box; the strains in the solid frame are PRV variant strains, and the other viruses The strain is a classic strain; the same sequence is represented by (.);

Fig. 3 is the specific primer and probe design position of amplifying Bartha-K61 vaccine strain;

Fig. 4 is the selection of DMSO concentration;

Fig. 5 is the amplification curve of triple fluorescent quantitative PCR; Wherein, A is the amplification curve of SC strain; B is the amplification curve of TJ strain; C is the amplification curve of Bartha-K61;

Figure 6 is the standard curve of triple fluorescent quantitative PCR; the logarithm of the standard concentration is the abscissa, and the CT value is the ordinate; wherein, A is the SC strain standard curve; B is the TJ strain standard curve; C is the Bartha-K61 standard curve;

Figure 7 shows the specificity of triple fluorescent quantitative PCR.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

Example 1 Establishment of triple fluorescent quantitative PCR method for differential detection of PRV classic strain, mutant strain and Bartha-K61 vaccine strain

1. Materials and methods

1.1 virus

Classical swine fever virus (CSFV) Shimen strain, porcine parvovirus (Porcine parvovirus, PPV) Z strain, porcine circovirus type 2 (Porcinecircovirus type 2, PCV2) JXL strain, porcine reproductive and respiratory syndrome virus (Porcinereproductiveandporcinerespiratorysyndromevirus, PRRSV) HuN4 strain , PRVSC strain, TJ strain, HLJMDJ2013 strain, HeBLP2014 strain, BJKJZ2015 strain (variant strain) and Bartha-K61 (vaccine strain) are preserved by the present inventor (wherein the pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain and BJKJZ2015 strain, by the present invention Human isolated, identified, preserved, and its partial sequence was uploaded to GenBank at the same time, the sequence accession numbers are KJ789182, KU670431, KU670430 and KU937306); Min-A strain (classic strain) was purchased from China Veterinary Drug Control Institute.

1.2 Virus genome extraction

The viral DNA in the tissue homogenate and cultured cells was repeatedly frozen and thawed at -80°C three times, and then extracted with a DNA extraction kit (Omega, USA).

1.3 Selection of primers and probes

NCBI website Blast analyzed the gene sequences of different PRV strains, designed multiple sets of public primer pairs and probes for the classic PRV strain and mutant strains at the gene deletion site of the Bartha-K61 vaccine strain, and designed multiple sets of primer pairs and probes for the sequence of the Bartha-K61 vaccine strain. Probes (Table 1, Table 2). At the same time, six combination schemes (Table 3) were designed to optimize the probes and primers. The experimental conditions were as follows: 0.5 μL each of the probe (10 μM); 1 μL each of the primers (10 μM); 1.5 μL each of the classical strain, mutant strain and Bartha-K61 genomic DNA; 10 μL of PremixExTaqProbeqPCR (TaKaRa, Japan); add water to each tube to 20 μL. The triple fluorescent quantitative PCR reaction program was set as follows: initial denaturation temperature, 95°C, 5min; denaturation temperature, 95°C, 35s, annealing and extension temperature, 60°C, 35s, 40 cycles; after each cycle, the fluorescence signal was detected. Select the experimental scheme by detecting the fluorescent signal. The CT value was defined as the number of cycles experienced when the amplification curve reached the threshold. Apply the selected primers and probes to subsequent experiments.

Table 1 Triple fluorescent quantitative PCR probe design sequence

Table 2 Triple fluorescent quantitative PCR primer design sequence

Table 3 Primer and probe combination scheme for triple fluorescent quantitative PCR

Note: a plus sign (+) indicates that the probe or primer was added in the protocol.

2. The selection results of triple fluorescent quantitative PCR primers and probes

The selection results of primers and probes showed that schemes 1 to 4 could not simultaneously detect and distinguish the classical strain, mutant strain and Bartha-K61 vaccine strain, while the application of scheme 5 could simultaneously detect and distinguish the three strains with the lowest CT value , the lowest detection limit and the highest sensitivity (Figure 1, Table 4). Therefore, the present invention uses the probes and primers in Scheme 5 (FAM-PRV-Cla (shown in SEQ ID No.1), HEX-PRV-Var (shown in SEQ ID No.4), Cy5-PRV-Bar (shown in SEQ ID No.6) shown), PRV-F1 (shown in SEQIDNo.8), PRV-R1 (shown in SEQIDNo.9), PRV-F2 (shown in SEQIDNo.14) and PRV-R2 (shown in SEQIDNo.15)) for subsequent During the experiment (Figure 2, Figure 3).

Table 4 Comparison of detection effects of triple fluorescent quantitative PCR using different combination schemes

The optimization of embodiment 2 triple fluorescence quantitative PCR method conditions

On the basis of the triple fluorescent quantitative PCR method established in Example 1, the present invention further optimizes the conditions of the triple fluorescent quantitative PCR.

1. Experimental method

Triple fluorescent quantitative PCR experiments were performed using the Mx3005p instrument (Stratagene, USA), and the concentrations of primers and probes were optimized by the value of the fluorescent signal (ΔRn). Experimental conditions are as follows: PCR reaction total volume is 20 μ L, FAM-PRV-Cla (shown in SEQIDNo.1), HEX-PRV-Var (shown in SEQIDNo.4) and Cy5-PRV-Bar (shown in SEQIDNo.6) ( 10 μM) each 0.5 μ L; PRV-F1 (shown in SEQIDNo.8), PRV-R1 (shown in SEQIDNo.9), PRV-F2 (shown in SEQIDNo.14) and PRV-R2 (shown in SEQIDNo.15) ( Genomic DNA 2 μL; PremixExTaqProbeqPCR (TaKaRa, Japan) 10 μL; meanwhile, 0%, 2.5%, 5% and 10% (v/v) dimethyl sulfoxide (DMSO) were added to the PCR reaction system, To screen the optimal concentration of DMSO. The triple fluorescent quantitative PCR reaction program was set as follows: initial denaturation temperature, 95°C, 5min; denaturation temperature, 95°C, 35s, annealing and extension temperature, 60°C, 35s, 45 cycles; after each cycle, the fluorescence signal was detected. The CT value of the sample exceeds the limit value and needs to be retested. The optimized experimental conditions and program settings were applied to subsequent experiments.

2. Experimental results

The results of DMSO condition optimization showed that the addition of DMSO can significantly improve the sensitivity of the method. Compared with the control group without adding DMSO, when the amount of DMSO added was 5% (v/v), the CT value was the lowest and the sensitivity was the highest (Figure 4, Table 5).

Table 5 Optimization of DMSO concentration

Specificity, sensitivity and repeatability test of embodiment 3 triple fluorescence quantitative PCR method

The optimized experimental conditions and program settings of triple fluorescent quantitative PCR in Example 2 were applied.

1. Experimental method

1.1 Standard curve establishment

Use primers PRV-F1/PRV-R1 to amplify the classic and mutant strains of PRV, use primers PRV-F2/PRV-R2 to amplify the Bartha-K61 vaccine strain, and clone the amplified products into pUC-19T In the vector, pUC-19T-SC, pUC-19T-TJ and pUC-19T-Bartha recombinant plasmids were constructed as detection standards for triple fluorescent quantitative PCR. The three recombinant plasmids were serially diluted 10 times from 5×10 ⁸ to 5 copies/μL, and stored at -20°C for future use.

The optimized triple fluorescent quantitative PCR detection method in Example 2 was used to detect the standard.

1.2 Specificity, sensitivity and repeatability tests

PK-15 cells were infected with PRVSC strain, TJ strain and Bartha-K61 vaccine strain respectively, and the virus genome was extracted, and detected by triple fluorescent quantitative PCR optimized in Example 2. Tests were also performed on CSFV, PCV2, PPV and PRRSV to test the specificity of the method. Subsequently, PK-15 cells were infected with PRVSC strain, TJ strain and Bartha-K61 vaccine strain respectively, and ten-fold serial dilution was carried out to test the sensitivity of the method. For PRVSC strain, TJ strain and Bartha-K61 vaccine strain, three repeated experiments with different titers were carried out, including within-group repetition and inter-group repetition, to test the repeatability of the method.

1.3 Triple fluorescent quantitative PCR method to identify different strains of PRV

The triple fluorescent quantitative PCR method was used to identify different strains of PRV, and the strains were sequenced at the same time. SC strain, TJ strain and Bartha-K61 vaccine strain were mixed in pairs or in threes, and the established method was used to detect the existence of virus mixture.

1.4 Virus isolation experiments to verify the triple fluorescent quantitative PCR method

The triple fluorescent quantitative PCR method was used to detect 234 samples from different provinces. All samples were rechecked with virus isolation assay and immunofluorescence assay, while positive samples were subjected to DNA sequencing.

2. Experimental results

2.1 Triple fluorescence quantitative PCR standard curve establishment

The standard product was detected by using the triple fluorescence quantitative PCR detection method established in the present invention (Fig. 5A-C). The logarithm of the concentration of the standard substance is the abscissa, and the CT value is the ordinate, and the standard curve of triple fluorescent quantitative PCR is established. The results showed that the standard curves of the SC strain and the TJ strain were in the range of 5×10 ⁸ to 5×10 ¹ copies, showing a linear relationship, and the standard curve of the Bartha-K61 strain was in the range of 5×10 ⁷ to 5 copies , showing a linear relationship (Fig. 6A-C). The linear regression equations of SC strain, TJ strain and Bartha-K61 strain were y=-3.436×log(x)+45.84, (R ² =0.995); y=-3.434×log(x)+43.93(R ² = 0.997); y = -3.054 x log(x) + 40.7, (R ² =0.995). Based on the detection range of this method, the limit values of SC strain, TJ strain and Bartha-K61 strain were 39.0, 38.1 and 38.5, respectively.

2.2 Specificity, sensitivity and repeatability of triple fluorescent quantitative PCR method

By detecting three fluorescent signals of FAM (specific to classic strains), HEX (specific to mutant strains) and Cy5 (specific to vaccine strains), this method can simultaneously distinguish SC strains, TJ strains and Bartha-K61 strains. The detection method established by the present invention was tested on CSFV, PCV2, PPV and PRRSV, and no fluorescent signal was detected, indicating that the method has strong specificity ( FIG. 7 ). The detection limits of the triple fluorescent quantitative PCR method for SC strain, TJ strain and Bartha-K61 were 0.5TCID ₅₀ , 0.2TCID ₅₀ and 0.05TCID ₅₀ , respectively. Repeatability tests within and between groups showed that the coefficient of variation of the method was less than 3%.

2.3 Triple fluorescent quantitative PCR method to identify different strains of PRV

The triple fluorescent quantitative PCR method was used to detect different strains of PRV. The results showed that this method could accurately identify different strains, and the identification results were the same as the sequencing results. At the same time, when PRVSC strains, TJ strains and Bartha-K61 vaccine strains are mixed in pairs or in threes, the method can also accurately identify the strain types in the virus mixture (Table 6).

Table 6 Triple fluorescent quantitative PCR to identify PRV strains

Note: a plus sign (+) indicates that the test result of the channel is positive;

bMinus sign (-) indicates that the test result of this channel is negative.

2.4 Validation of triplex quantitative PCR method with virus isolation

234 samples were detected by triple fluorescent quantitative PCR method, and the results showed that 8 samples contained PRV classic strain; 34 samples contained variant strain; 6 samples contained Bartha-K61 strain. The results of virus isolation showed that 8 samples contained the classic strain of PRV; 35 samples contained the mutant strain; 6 samples contained the Bartha-K61 strain. The coincidence rates of the two methods for the detection of classic strains, variant strains and vaccine strains were 100%, 99.5% and 100% respectively (Table 7). The DNA sequencing results of positive samples showed that the triple fluorescent quantitative PCR method can accurately identify different strains of PRV.

Table 7 Compatibility of triple fluorescent quantitative PCR and virus isolation

Claims (10)

1. for differentiating the triple fluorescent quantitative PCR's of pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain Primer and probe, it is characterised in that described primer includes: nucleotide sequence shown in SEQ ID No.8 and SEQ ID No.9 forms Primer to 1, shown in SEQ ID No.10 and SEQ ID No.11 the primer of nucleotide sequence composition to 2, SEQ ID No.12 With the primer of the composition of nucleotide sequence shown in SEQ ID No.13 to any pair in 3, and SEQ ID No.14 and SEQ The primer of the composition of nucleotide sequence shown in ID No.15 is to 4；

Described probe includes: any one in nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 Kind, any one in nucleotide sequence shown in SEQ ID No.4 or SEQ ID No.5, and SEQ ID No.6 or SEQ ID Any one in nucleotide sequence shown in No.7.

2. according to the primer described in claim 1 and probe, it is characterised in that described primer includes: SEQ ID No.8 and SEQ The primer of the composition of nucleotide sequence shown in ID No.9 is to 1, and nucleotides sequence shown in SEQ ID No.14 and SEQ ID No.15 The primer of row composition is to 4；

Described probe includes: nucleotide sequence shown in SEQ ID No.1, SEQ ID No.4 and SEQ ID No.6.

3. according to the primer described in claim 1 and probe, it is characterised in that: nucleotides sequence is classified as SEQ ID No.1, SEQ ID 5 ' ends of the probe shown in No.2 or SEQ ID No.3 are marked with FAM fluorescent reporter group respectively, and 3 ' ends are marked with BHQ1 respectively Fluorescent quenching group；

Nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.4 or SEQ ID No.5 and is marked with HEX fluorescence report respectively Group, 3 ' ends are marked with BHQ1 fluorescent quenching group respectively；

Nucleotides sequence is classified as 5 ' ends of the probe shown in SEQ ID No.6 or SEQ ID No.7 and is marked with Cy5 fluorescence report respectively Group, 3 ' ends are marked with BHQ3 fluorescent quenching group respectively.

4. according to the primer described in claim 1 and probe, it is characterised in that described pseudorabies virus classics strain includes: pseudo- Rabies virus SC strain or Min-A strain；Described variant includes: pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain or BJKJZ2015 strain.

5. primer described in claim 1 or 2 and probe preparation differentiate pseudorabies virus classics strain, variant and Application in the reagent of Bartha-K61 vaccine strain or material.

6. according to the application described in claim 5, it is characterised in that: described pseudorabies virus classics strain includes: pseudorabies Virus SC strain or Min-A strain；Described variant includes: pseudorabies virus TJ strain, HLJMDJ2013 strain, HeBLP2014 strain or BJKJZ2015 strain.

7. the triple fluorescent quantitative PCR inspection differentiating pseudorabies virus classics strain, variant and Bartha-K61 vaccine strain Test agent box, including: primer, probe, Premix Ex Taq Probe qPCR and distilled water；It is characterized in that, described primer It is the primer described in claim 1 or 2 and probe with probe.

8. according to the triple fluorescent quantitative PCR detection kit described in claim 7, it is characterised in that also include that dimethyl is sub- Sulfone.

9. according to the triple fluorescent quantitative PCR detection kit described in claim 8, it is characterised in that described dimethyl sulfoxide Consumption be by primer, probe, Premix Ex Taq Probe qPCR, distilled water, testing gene group DNA and dimethyl sulfoxide The 0%-10%, preferably 2.5%-10% of the triple fluorescent quantitative PCR reaction system cumulative volume of composition.

10. according to the triple fluorescent quantitative PCR detection kit described in claim 9, it is characterised in that described dimethyl sulfoxide Consumption be by primer, probe, Premix Ex Taq Probe qPCR, distilled water, testing gene group DNA and dimethyl sulfoxide The 5% of the triple fluorescent quantitative PCR reaction system cumulative volume of composition.