CN106048079B

CN106048079B - Double-color fluorescence detection method, primer and probe for rapidly distinguishing PRV Chinese type, European and American type and vaccine strains

Info

Publication number: CN106048079B
Application number: CN201610296853.3A
Authority: CN
Inventors: 张建峰; 刘志成; 孙俊颖; 张春红; 沈海燕
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2016-05-05
Filing date: 2016-05-05
Publication date: 2020-03-31
Anticipated expiration: 2036-05-05
Also published as: CN106048079A

Abstract

The invention discloses a two-color fluorescence detection method, primers and probes for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61. The method combines a real-time fluorescence PCR technology and a melting curve analysis technology, and identifies the Chinese type, the European and American type and the vaccine strain Bartha-K61 of the porcine pseudorabies virus according to the Tm value difference of the melting curve; the operation is simple: only one reaction is needed to realize the identification and detection of two genotypes and a vaccine strain Bartha-K61; the detection speed is high and the flux is high: the whole operation process can be completed within 3 hours, cell culture of the virus is not needed, and the time for identifying and detecting the Chinese type, the European and American type and the vaccine strain Bartha-K61 of the porcine pseudorabies virus is greatly shortened; the method has the advantages of high accuracy, good specificity and good repeatability, can accurately, quickly and highly-flux analyze, and is favorable for popularization and application in clinical practice.

Description

Double-color fluorescence detection method, primer and probe for rapidly distinguishing PRV Chinese type, European and American type and vaccine strains

Technical Field

The invention relates to a method for identifying different genotypes of viruses, vaccine viruses and wild strains, in particular to a two-color fluorescence detection method and a kit for rapidly distinguishing Chinese, European and American porcine Pseudorabies viruses (PRV) and vaccine strains Bartha-K61, and the method is a probe melting curve analysis technology based on two double-labeled self-quenching probes and is used for detecting the Chinese, European and American porcine Pseudorabies viruses and the vaccine strains Bartha-K61.

Background

2011 since China, large-scale pig farms such as North China, China and south China have the pseudorabies outbreak after the immunization of the pseudorabies vaccine, the wild virus antibody positive rate of part of pig farms is up to 100 percent, and the pregnancy is pregnant35 percent of sows have abortion, which is a great impact encountered in the pig industry of domestic pigs. The vaccination is an effective means for controlling the porcine pseudorabies at present, and at present, 4 PRV commercial attenuated vaccines (Bartha-K61 strain, Bucharest strain, HB-98 strain and SA215 strain) are clinically used in China, wherein the Bartha-K61 strain has higher market share. There are 1 additional commercial inactivated vaccines: the Esche A strain is also marketed. The inactivated vaccine is higher than the attenuated vaccine in the aspect of safety, but cannot be proliferated in pseudorabies virus latent tissues (such as trigeminal nerve and the like), and cannot play a role in preventing the pigs with latent infection; the porcine pseudorabies gene deletion vaccine has weaker toxicity and better immunogenicity, but has the danger of virus dispersion caused by epidemic disease and latent infection due to virulence reversion, so a matched vaccine detection method is necessary to be established. In 2015, it was confirmed by the phylogenetic analysis of PRV whole gene and 67 genes that PRV can be divided into two evolutionary branches: european and American type (Genotype I, found in Europe, America, Australia, Asia, etc.) and Chinese type (Genotype II, found in China, Malaysia, etc.), representative strains of European and American type are Bartha, Becker, Kaplan, HS, etc.; representative strains of Chinese type include Fa, Ea, HeN1, JS, TJ, etc. (Chao Ye,etal, &,Guang-Zhi Tong. Virology (2015) 483: 32-43). The epidemic strain of porcine pseudorabies virus is relatively close to the Chinese strains Fa and Ea at present (Xiuling Yu,etal, &,Kegong Tian. Emerging Infectious Diseases (2014) 20: 102-104；Yinbiao Wang,etal, &,Gaiping Zhang.virus Genes (2015) 50: 401-409) and the need to track and detect whether European and American porcine pseudorabies viruses appear clinically, so the establishment of the identification and detection method for the Chinese and European and American porcine pseudorabies viruses is significant.

Although the combined use of the gpI (gE) antibody ELISA detection kit and the gB antibody ELISA detection kit which are established according to the deletion of all or part of gE genes of the gene-deleted vaccine can achieve the aim of identifying vaccine virus infection, the antibody detection has serious hysteresis, and simultaneously the type of a strain cannot be identified, so that the further guidance of vaccine immunity is not facilitated, so that a method which is relatively simple and easy to operate, reliable in detection result, high in flux and rapid in distinguishing different genotypes and vaccine strains of the porcine pseudorabies virus is urgently needed at present.

Disclosure of Invention

In order to solve the problems, the invention establishes a two-color fluorescence PCR detection method for rapidly distinguishing Chinese type, European and American type of the porcine pseudorabies virus and a vaccine strain Bartha-K61, and the method has the advantages of simple and rapid operation, high flux and reliable detection result, and is favorable for popularization and application in clinical practice.

The invention aims to provide a double-color fluorescent PCR primer and a probe for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and a vaccine strain Bartha-K61.

The invention also aims to provide a two-color fluorescence PCR method for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61.

The invention further aims to provide a double-color fluorescent PCR kit for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61.

The technical scheme adopted by the invention is as follows:

a double-color fluorescent PCR primer for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and a vaccine strain Bartha-K61 has the following nucleotide sequence:

and (3) primer F: 5'-GGGCGTCTACACGTGGCGC-3' (SEQ ID NO: 1),

and (3) primer R: 5'-GTTGGTCACGAAGGCGGCGT-3' (SEQ ID NO: 2).

A double-color fluorescent PCR probe for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61 has the following nucleotide sequence:

probe P1: 5'-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-3' (SEQ ID NO: 3),

probe P2: 5'-CCGAGTTCGGCCTGAGCGCGCCGCC-3' (SEQ ID NO: 4).

Furthermore, the fluorescent group marked at the 5 'end of the probe sequence is one of FAM, HEX, VIC, CY5 and TET, and the quenching group marked at the 3' end of the probe sequence is one of TAMRA, MGB and BHQ; and the 5' -end labeled fluorescent groups of the probes P1 and P2 are different.

A double-color fluorescent PCR kit for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and a vaccine strain Bartha-K61 contains the primer.

Further, the kit also contains the probe.

A double-color fluorescence PCR method for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and a vaccine strain Bartha-K61 comprises the following steps:

1) extracting viral DNA from the sample;

2) performing a fluorescent PCR amplification reaction by using the extracted DNA as a template and the primer pair F, R, the probe P1 and the probe P2 to obtain an amplification product;

3) and (4) carrying out melting curve analysis on the amplification product to determine the virus type in the sample.

Further, the fluorescent PCR amplification reaction system in the step 2) is as follows:

Premix Ex-Taq 5.0μl

primer P1 (1. mu.M) 0.4. mu.l

Primer P2 (10. mu.M) 0.4. mu.l

Probe1 (10. mu.M) 0.2. mu.l

Probe2 (10. mu.M) 0.2. mu.l

Template 1.0. mu.l

ddH₂O 2.8μl。

Further, the fluorescent PCR amplification reaction procedure in the step 2) is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle was 55 times.

Further, the melting curve analysis program in step 3) is: denaturation at 95 ℃ for 10 sec; melting curve analysis was performed by continuously collecting fluorescence signals of probes P1 and P2 at 5/deg.C at a rate of 0.13 deg.C/s from 40 deg.C to 97 deg.C.

Further, the specific analysis process of the melting curve analysis in step 3) is as follows:

1) in the fluorescence detection result of the probe P1, when the Bartha-K61 standard sample is used as a control, if the absolute value of the Δ Tm value of the melting temperature of the sample and the melting temperature of the positive control Bartha-K61 is less than 1.0 ℃, the swine pseudorabies virus is judged to be European and American;

2) in the fluorescence detection result of the probe P1, when an HDDJ standard sample is used as a control, if the absolute value of the Tm value of the melting temperature of the sample and the melting temperature of the positive control HDDJ is less than 1.0 ℃, the sample is judged as the Chinese porcine pseudorabies virus;

3) in the fluorescence detection result of the probe P2, when the Bartha-K61 standard sample is used as a control, if the absolute value of the Δ Tm value of the melting temperature of the sample and the melting temperature of the positive control Bartha-K61 is less than 1.0 ℃, the test is judged as the Bartha-K61 vaccine strain.

The invention has the beneficial effects that:

1) the invention establishes a two-color fluorescence detection method, primers and probes for rapidly distinguishing Chinese type, European and American type of porcine pseudorabies virus and vaccine strain Bartha-K61 for the first time. The operation is simple: only one reaction is needed to realize the identification and detection of Chinese type, European and American type and vaccine strain Bartha-K61; the detection speed is high and the flux is high: the whole operation process can be completed within 3 hours, cell culture of the virus is not needed, and the time for identifying and detecting the Chinese type and European and American type strains of the porcine pseudorabies virus and the Bartha-K61 vaccine strain is greatly shortened; the method has the advantages of high accuracy, good specificity and good repeatability, can accurately, quickly and highly-flux analyze, and is favorable for popularization and application in clinical practice.

2) The PCR primer pair F/R can specifically amplify the porcine pseudorabies virus wild virus Chinese type, European and American type and the vaccine strain Bartha-K61, is beneficial to improving the PCR efficiency and reducing the virus identification and typing time. The probe P1 can be specifically hybridized with Chinese type and European and American type nucleic acid sites of the porcine pseudorabies virus, and has better specificity. The probe P2 can be specifically hybridized with identification sites of porcine pseudorabies virus Bartha-K61 vaccine strains and other non-Bartha-K61 strains, and has better specificity. The primers F/R, the probes P1 and P2 are not combined with other common porcine reproductive disorder related virus nucleic acids, so that the accuracy of the result analysis is improved.

4) The lowest detection limit of the double-color fluorescence detection method for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61 can reach single copy, and the sensitivity is higher.

Drawings

FIG. 1 is a positive standardized sample gene evolution analysis diagram, wherein ● is standardized sample Chinese type HDDJ, &lTtTtransformation = a &gTt &lTt/T &gTtis standardized sample Chinese type Ea, ■ is standardized sample Chinese type Fa, ◇ is standardized sample Bartha-K61 vaccine strain (also European and American type), △ is standardized sample European and American type HS;

FIG. 2 is a melting curve diagram of a standardized sample dual-color fluorescence detection method, wherein A is a melting curve diagram for FAM and HEX dual-channel simultaneous detection, and B and C are melting curve diagrams for FAM and HEX single-channel separate detection; the standard sample is Bartha-K61 vaccine strain (also European and American type), non-Bartha-K61 strain HDDJ (also Chinese type);

FIG. 3 is a melting curve diagram of a specificity test of a two-color fluorescence detection method, wherein A is a melting curve diagram for FAM and HEX double-channel simultaneous detection, and B and C are melting curve diagrams for FAM and HEX single-channel detection respectively;

FIG. 4 is a graph showing the amplification curve and the melting curve of a sensitivity test for a positive plasmid p-Bartha-K61 in a two-color fluorescence detection method, wherein A is a graph showing the amplification curve of simultaneous detection of FAM and HEX channels, B is a graph showing the melting curve of simultaneous detection of FAM and HEX channels, and C and D are graphs showing the amplification curve of separate detection of FAM single channel and HEX single channel; e and F are respectively FAM single channel and HEX single channel and respectively detect a melting curve diagram;

FIG. 5 is a graph showing the amplification curve and the melting curve of the positive plasmid p-HDDJ sensitivity test in the two-color fluorescence detection method, wherein A is a FAM and HEX two-channel simultaneous detection amplification curve, B is a FAM and HEX two-channel simultaneous detection melting curve, and C and D are FAM single-channel and HEX single-channel respective detection amplification curves; e and F are respectively FAM single channel and HEX single channel and respectively detect a melting curve diagram;

FIG. 6 is a melting curve diagram of a clinical sample dual-color fluorescence detection method, wherein A is a melting curve diagram for FAM and HEX dual-channel simultaneous detection, and B and C are melting curve diagrams for FAM and HEX single-channel separate detection; the standard samples Bartha-K61 vaccine strain (also European and American) and non-Bartha-K61 strain HDDJ (also Chinese) are positive controls, and 4 clinical samples are numbered as INT, S, ZZ and LC respectively; the results show that samples INT and S are European and American strains, and ZZ and LC are Chinese strains, which are not Bartha-K61 vaccine strains. The results have been verified by sequencing.

Detailed Description

The present invention will be further described with reference to the following examples, but is not limited thereto.

Example 1 primers and probes

After screening a large number of designed primers and probes, the primer pair F, R, the probes P1 and P2 have the best effect of distinguishing Chinese, European and American porcine pseudorabies viruses and the vaccine strain Bartha-K61 by a two-color fluorescence method, and the base sequences are shown as follows.

And (3) primer F: 5'-GGGCGTCTACACGTGGCGC-3' (SEQ ID NO: 1),

and (3) primer R: 5'-GTTGGTCACGAAGGCGGCGT-3' (SEQ ID NO: 2),

probe P1: 5 '-FAM-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-BHQ 1-3' (SEQ ID NO: 3),

probe P2: 5 '-HEX-CCGAGTTCGGCCTGAGCGCGCCGCC-BHQ 1-3' (SEQ ID NO: 4).

Example 2 preparation of Standard samples, two-color fluorescent PCR amplification and melting Curve analysis

1) Extracting porcine pseudorabies virus DNA:

respectively taking pathological material samples suspected to be infected with PRV, wherein the pathological material samples can be lymph nodes, brain, heart, liver, spleen, lung, kidney, tonsil and other tissues of pigs died of diseases, or collected pig serum or nasal type seeds. The serum can be directly taken 200 mul for standby; dissolving the seeds in 1mL PBS hydrochloric acid buffer solution, standing for 10-20min, and collecting 200 μ l; grinding a tissue sample of a dead pig, centrifuging and taking 200 mu l of supernatant for later use; Bartha-K61 vaccine was dissolved in 3mL PBS HCl buffer, and 200. mu.L of the solution was taken for use. Nucleic acid Extraction was performed as described in the MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 of TAKARA.

2) Preparation of standard sample:

in order to verify the feasibility and reliability of the method, construct a standard positive sample (which is determined to be correct by sequence determination) and provide a positive control for the detection of a subsequent clinical sample, the method needs to prepare a positive standard sample of the porcine pseudorabies virus Bartha-K61 vaccine strain (which is European and American), a European strain HS, a Chinese strain HDDJ, a Chinese strain Fa and a Chinese strain Ea. The preparation steps of the standard sample are as follows: Bartha-K61 vaccine strain (European and American type), European strain HS, Chinese strain HDDJ, Chinese strain Fa and Chinese strain Ea are taken to be respectively subcultured in ST cells (pig testicular cells) until the state is stable, and 200 mu l of virus liquid is taken to extract nucleic acid as a positive standard substance according to the extraction method of the DNA of the porcine pseudorabies virus.

By the method, the porcine pseudorabies virus Bartha-K61 vaccine strain (also European and American), European strain HS, Chinese strain HDDJ, Chinese strain Fa and Chinese strain Ea containing the target gene segment are respectively obtained.

To verify the genotype of the selected standard positive samples, the samples were analyzed for genotype as reported in the literature (Chao Ye,etal, &,Guang-Zhi Tong. Virology (2015) 483: 32-43), and selecting corresponding genes with typing characteristics for evolutionary tree analysis, as shown in FIG. 1.

FIG. 1 is a gC gene evolution analysis diagram of a standardized sample, wherein ● is a standardized sample Chinese type HDDJ, &lTtTtransformation = a &gTtL &lTt/T and gTt is a standardized sample Chinese type Ea, ■ is a standardized sample Chinese type Fa, ◇ is a standardized sample Bartha-K61 vaccine strain (also in European and American types), △ is a standardized sample European and American type HS. from FIG. 1, the Bartha-K61 vaccine strain, HS are European and American types (Genotype I), and HDDJ, Fa, Ea are Chinese types (Genotype II).

3) The fluorescent PCR operation steps of the positive standard sample are as follows:

respectively taking the three obtained positive standard samples as DNA templates, and respectively carrying out fluorescence PCR amplification reaction and melting curve analysis;

and (3) PCR reaction system:

ddH₂O 2.8μl

Premix Ex-Taq 5.0μl

mu.l of 1. mu.M primer F0.4. mu.l

10 μ M primer R0.4 μ l

10μM P1 0.2μl

10μM P2 0.2μl

Template 1.0. mu.l

The total volume was 10. mu.l.

The PCR amplification reaction procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle was 55 times.

The melting curve analysis program was as follows:

denaturation at 95 ℃ for 10 sec; FAM and HEX fluorescence signals were collected continuously at a rate of 0.13 ℃/s at 40 ℃ to 97 ℃ for 5/DEG C and subjected to melting curve analysis.

4) Analysis of melting curve analysis result of positive standard sample

The PCR amplification products were analyzed using a LightCycler 96 Analyzer. The melting curve analysis results of 5 positive standard samples of porcine pseudorabies virus are shown in figure 2.

FIG. 2 is a melting curve diagram of a double-color fluorescence detection method for a positive standard sample, and FIG. 2A is a superimposed graph of fluorescence curves of FIGS. 2B and 2C.

FIG. 2B shows the FAM fluorescence channel analysis result (i.e., the fluorescence detection result of probe P1), from which it can be seen that the melting curves of European and American strains Bartha-K61, HS and Chinese strains HDDJ, Fa, Ea standard samples are separated from each other, indicating that the designed primer F, R and probe P1 are suitable for the melting curve analysis of the Chinese and European and American genotypes of porcine pseudorabies virus. According to the difference of melting temperatures (Tm) of two positive standard samples, the European and American strain Bartha-K61 has a lower melting temperature of 70.42 +/-0.15 ℃ and the Chinese strain HDDJ has a higher melting temperature of 76.8 +/-0.07 ℃ (FIG. 2A and FIG. 2B).

FIG. 2C shows the analysis result of HEX fluorescence channel (i.e. the fluorescence detection result of probe P2), from which it can be seen that the melting curves of vaccine strain Bartha-K61 and non-Bartha-K61 strain HS, HDDJ, Fa, Ea standard samples are separated from each other, indicating that the designed primer F, R and probe P2 are suitable for the melting curve analysis of porcine pseudorabies virus vaccine strain Bartha-K61 and non-Bartha-K61 strain. According to the difference of the melting temperatures (Tm) of two standard samples, the melting temperature of the vaccine strain Bartha-K61 is lower than 71.66 ℃, and the melting temperature of the non-Bartha-K61 strain is higher than 76.08 +/-0.08 ℃ (FIG. 2A and FIG. 2C).

Example 3 specificity test

The detection method established in the present invention is specifically detected as follows.

Other viral nucleic acids commonly associated with Porcine reproductive and Respiratory Syndrome are extracted separately, such as Porcine pestivirus (CSFV), Porcine encephalitis B Virus (JEV), Porcine circovirus type 2 (PCV 2), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Parvovirus (PPV), cDNA reverse transcription of CSFV, JEV and PRRSV, cDNA of the above facial Virus, PCV 2 and PPV nucleic acids and water as PCR templates, respectively, by PCR amplification and melting curve analysis in (2), and compared with Bartha-K61 strain (also European vaccine type) and non-Bartha-61 strain (also called HDJ-61 strain), as shown in FIG. 3, and as a standard melting curve, as shown in FIG. 3.

As can be seen from FIGS. 3A-C, the detection method of the invention can only specifically amplify the melting peak of the positive standard sample of the porcine pseudorabies Bartha-K61 vaccine strain (also European and American) and the non-Bartha-K61 strain HDDJ (also Chinese), while other porcine reproductive disturbance disease related viruses such as CSFV, JEV, PRRSV, PCV 2 and PPV do not amplify the specific melting peak. The primer F, R and the probes P1 and P2 are shown to have better specificity, and can be used for the fluorescent detection of Chinese and European types of the porcine pseudorabies virus and vaccine strains Bartha-K61.

Example 4 sensitivity test

The detection method established in the present invention was subjected to sensitivity detection as follows.

Taking the standard product Bartha-K61 in example 2 as a template and the primers P1 and P2 as amplification primers, cloning the amplification product into PMD18T-Vector, constructing positive plasmid P-Bartha-K61, carrying out nucleic acid detection on P-Bartha-K61, converting the number of the plasmids, and carrying out 10-fold gradient dilution to form 1.0x10⁹、1.0x10⁸、1.0x10⁷、1.0x10⁶、1.0x10⁵、1.0x10⁴、1.0x10³、1.0x10²、1.0x10¹、1.0x10⁰The fluorescence PCR amplification reaction and melting curve analysis method in example 2 were performed using 10 gradients of copies/. mu.l, and the amplification profile and melting curve peaking profile are shown in FIG. 4.

FIG. 4 is a graph of the amplification curve and the melting curve of the two-color fluorescence detection method for positive plasmid p-Bartha-K61, wherein A is the amplification curve of the FAM and HEX two-channel simultaneous detection, i.e., the superimposed graph of the curves in the C and D graphs; c and D are amplification curve graphs of FAM single-channel detection and HEX single-channel detection respectively; b is a melting curve chart of FAM and HEX double-channel simultaneous detection, namely an overlay graph of curves in E and F graphs; e and F are respectively melting curve graphs of FAM single-channel detection and HEX single-channel detection. It can be seen that the detection method shows a obviously reduced fluorescence signal along with the reduction of the concentration of the nucleic acid, the number of the plasmids is as low as 1.0 copy/microliter, and the FAM channel and the HEX channel can also detect the corresponding fluorescence signals.

Positive plasmid p-HDDJ was constructed in the same manner as described above, diluted 10-fold in gradient, and analyzed by the fluorescence PCR amplification reaction and melting curve analysis method described in example 2 above, and the amplification profile and melting curve peaking chart are shown in FIG. 5.

FIG. 5 is a graph of the amplification curve and the melting curve of the double-color fluorescence detection method for positive plasmid p-HDDJ, wherein A is the amplification curve of the FAM and HEX double-channel simultaneous detection, i.e., the superimposed graph of the curves in the C and D graphs; c and D are amplification curve graphs of FAM single-channel detection and HEX single-channel detection respectively; b is a melting curve chart of FAM and HEX double-channel simultaneous detection, namely an overlay graph of curves in E and F graphs; e and F are respectively melting curve graphs of FAM single-channel detection and HEX single-channel detection. It can be seen that the detection method shows a obviously reduced fluorescence signal along with the reduction of the concentration of the nucleic acid, the number of the plasmids is as low as 1.0 copy/microliter, and the FAM channel and the HEX channel can also detect the corresponding fluorescence signals.

The detection results show that the method has higher sensitivity.

Example 5 fluorescent PCR amplification and melting Curve analysis of clinical samples

1) Extraction of viral nucleic acids from samples: the method is the same as the nucleic acid extraction method in the above example 2, and viral nucleic acid in 4 clinical samples is extracted;

2) the method is the same as the fluorescence PCR amplification reaction and the melting curve analysis in the above example 2 by using the extracted viral nucleic acid as a template; meanwhile, the positive standard substance porcine pseudorabies virus Bartha-K61 vaccine strain (which is also European and American) and non-Bartha-K61 strain HDDJ (which is also Chinese) which are described in the example 2 are used as positive controls.

3) Analysis of results of melting curve analysis of clinical samples

The fluorescent PCR amplification products were analyzed using a LightCycler 96 Analyzer. 4 clinical samples were tested, and the results of melting curve analysis are shown in FIG. 6.

As can be seen from the melting curve chart of the clinical sample bicolor fluorescence detection method in FIG. 6, when the melting curves of PRV European and American Bartha-K61 and Chinese HDDJ standard sample are used as controls in analyzing the FAM fluorescence channel (i.e., the detection result of the probe P1), the sample to be detected and the positive Bartha-K61 are judged as European and American PRV when the absolute value of the melting peak Tm value is less than 1.0 ℃; judging the sample to be detected as Chinese type PRV when the absolute value of the melting peak Δ Tm between the sample to be detected and the positive control HDDJ is less than 1.0 ℃; the results showed that the numbers INT and S in the 4 clinical samples tested were European and American type PRV, and the numbers ZZ and LC were Chinese type PRV (see FIGS. 6A and B).

When an HEX fluorescence channel (namely the detection result of the probe P2) is analyzed, when the melting curve of the PRV vaccine strain Bartha-K61 and the HDDJ standard sample of the non-Bartha-K61 strain are used as a control, the PRV vaccine strain Bartha-K61 is judged when the absolute value of the melting peak Tm value between the sample to be detected and the positive control Bartha-K61 is less than 1.0 ℃; judging the non-PRV vaccine Bartha-K61 when the absolute value of the melting peak Δ Tm between the sample to be detected and the positive control HDDJ is less than 1.0 ℃; the results showed that none of INT, S, ZZ and LC were detected in 4 clinical samples as PRV vaccine strain Bartha-K61.

By combining the FAM channel and HEX channel results (namely the detection results of the probes P1 and P2), the numbers INT and S in 4 clinical samples are European and American type PRV, ZZ and LC are Chinese type PRV, and the samples are not PRV vaccine strain Bartha-K61. The results have been verified by sequencing.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

<110> institute of animal health of academy of agricultural sciences of Guangdong province

<120> two-color fluorescence detection method, primer and probe for rapidly distinguishing PRV (porcine reproductive and respiratory syndrome) Chinese type, European and American type and vaccine strains

Needle

<130>

<160>4

<170>PatentIn version 3.5

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<213> Artificial sequence

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gggcgtctac acgtggcgc 19

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gttggtcacg aaggcggcgt20

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cgcagcgcca acgtctcgct cgtcctgtac 30

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ccgagttcgg cctgagcgcg ccgcc 25

Claims

1. A double-color fluorescent PCR primer and probe for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61 has the nucleotide sequences as follows:

and (3) primer F: 5'-GGGCGTCTACACGTGGCGC-3' (SEQ ID NO: 1);

and (3) primer R: 5'-GTTGGTCACGAAGGCGGCGT-3' (SEQ ID NO: 2);

probe P1: 5'-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-3' (SEQ ID NO: 3);

probe P2: 5'-CCGAGTTCGGCCTGAGCGCGCCGCC-3' (SEQ ID NO: 4).

2. The primer and probe as claimed in claim 1, wherein the fluorescent group labeled at 5 'end of the probe sequence is one of FAM, HEX, VIC, CY5 and TET, and the quencher labeled at 3' end of the probe sequence is one of TAMRA, MGB and BHQ; and the 5' -end labeled fluorescent groups of the probes P1 and P2 are different.

3. A two-color fluorescent PCR kit for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61, which is characterized in that the kit contains the primer and the probe in claim 1 or 2.

4. A non-diagnostic double-color fluorescence PCR method for rapidly distinguishing Chinese type, European and American type porcine pseudorabies virus and vaccine strain Bartha-K61 is characterized by comprising the following steps:

s01, extracting virus DNA from a sample;

s02, performing a fluorescence PCR amplification reaction by using the extracted DNA as a template and using the primer pair F and R as claimed in claim 1, and the probe P1 and the probe P2 as claimed in claim 1 or 2 to obtain an amplification product;

s03, carrying out melting curve analysis on the amplification product to determine the virus type in the sample;

the specific analysis process of the melting curve analysis is as follows:

s11, in the fluorescence detection result of the probe P1, a Bartha-K61 standard sample is used as a control, and if the absolute value of the delta Tm value of the melting temperature of the sample and the melting temperature of a positive control Bartha-K61 is less than 1.0 ℃, the porcine pseudorabies virus in Europe and America is judged;

s12, in the fluorescence detection result of the probe P1, taking an HDDJ standard sample as a control, and if the absolute value of the delta Tm value of the melting temperature of the sample and the melting temperature of the positive control HDDJ is less than 1.0 ℃, determining the sample as the Chinese porcine pseudorabies virus;

s13, in the fluorescence detection result of the probe P2, a Bartha-K61 standard sample is used as a control, and if the absolute value of the delta Tm value of the melting temperature of the sample and the melting temperature of a positive control Bartha-K61 is less than 1.0 ℃, the Bartha-K61 vaccine strain is judged.

5. The method of claim 4, wherein: the fluorescent PCR amplification reaction system in the step S02 is as follows:

6. the method of claim 4, wherein: the fluorescent PCR amplification reaction procedure in step S02 is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle was 55 times.

7. The method of claim 4, wherein: the melting curve analysis program in step S03 is: denaturation at 95 ℃ for 10 s; melting curve analysis was performed by continuously collecting fluorescence signals of probes P1 and P2 at 5/deg.C at a rate of 0.13 deg.C/s from 40 deg.C to 97 deg.C.