CN113136456A - Fluorescent quantitative PCR detection kit for identifying porcine pseudorabies virus gene deletion vaccine strain and wild strain - Google Patents

Abstract

The invention relates to the field of animal plague detection, in particular to a fluorescent quantitative PCR detection kit for identifying a porcine pseudorabies virus gene deletion vaccine strain and a wild strain. In the detection kit provided by the invention, primers and probes for detecting the gI/gE gene deletion condition of the porcine pseudorabies virus are shown in SEQ ID NO. 1-3. The detection kit provided by the invention also comprises a hot start DNA polymerase modified based on a chemical method, a dUTP/UNG anti-pollution system and an anti-PCR inhibitor factor. The detection kit provided by the invention has high sensitivity, and the lowest detection limit is 189 copies/uL. The kit is suitable for detecting complex animal clinical samples.

Description

Fluorescent quantitative PCR detection kit for identifying porcine pseudorabies virus gene deletion vaccine strain and wild strain

Technical Field

The invention relates to the field of animal plague detection, in particular to a fluorescent quantitative detection kit for identifying gI/gE gene deletion vaccine strains and wild strains of PRV.

Background

Pseudorabies (PR or Aujeszky's disease, AD) is an acute, febrile infectious disease of many domestic and wild animals caused by infection with Pseudorabies virus (PRV). Pseudorabies virus can cause the pigs to generate subclinical infection and latent infection, but the clinical symptoms are different due to different ages in days, the piglets often show symptoms such as high fever, loss of appetite, salivation, dyspnea, tremor, diarrhea and the like, and then have dyskinesia, intermittent convulsion and coma until death (the death rate of the piglets within 15 days is often up to 100 percent, and the death rate of weaned piglets is 10 to 20 percent); the fattening pigs only show slight respiratory symptoms, slow weight gain and the like; the sows show return of emotion and are frequently bred to be infertile, and pregnant sows often show abortion, stillbirth and mummy; boars often suffer from testicular swelling, atrophy, etc., and have reduced or lost fertility.

PRV is a highly resistant herpesvirus, and has a variety of proteins, among which envelope component proteins gB and gC induce cellular and humoral immune responses, and gE is a major virulence protein capable of promoting fusion of infected and uninfected cells, and is an essential pathogenic factor for virus invasion of the central nervous system. Because the gE gene-deleted attenuated vaccine is safer than the PRV vaccine in which other genes are deleted alone, only the gE gene-deleted vaccine is allowed to be used in European Union and the United states at present. At present, more than 1200 wild pseudorabies strains all contain gE genes, so that the existence of a gE protein antibody is an important basis for identifying wild viruses and vaccine strains. It is presently believed that the gE and gI proteins together form a non-covalently associated complex that is present in infected cell membranes and viral capsids and that plays a role in the invasion and spread of PRV in the nervous system, but that the gE and gI proteins do not have a common antigenic determinant.

The Quantitative Real-time PCR (Quantitative Real-time PCR, qPCR) technology is that a fluorescent group is added into a PCR reaction system, the whole PCR process is monitored in Real time by using fluorescent signal accumulation, and finally, an unknown template is quantitatively analyzed through a standard curve. Real-time fluorescence quantitative PCR has been widely used in various fields of molecular biology research as an extremely effective experimental method. Compared with the conventional PCR, the technology can rapidly and sensitively detect the viral RNA and DNA and the bacterial DNA.

Disclosure of Invention

The invention aims to provide a method for identifying and distinguishing gI/gE gene deletion strains and wild strains of PRV. In particular, a method for identifying and distinguishing PRV wild strains from HB98 and HB2000 gene deletion strains is provided.

HB98 and HB2000 of the department of academic, vocational and academic institutions are vaccines with partial deletion of gI and gE genes of PRV, and the existing fluorescent quantitative detection kit in the market cannot detect HB98 and HB2000(gI and gE gene deletion) vaccine strains of the department of academic, vocational and academic institutions, possibly because the deleted nucleotide sequences of the two vaccine strains are relatively few and the risk of virus variation objectively exists. The invention establishes the identification and detection method of the wild strain and the gene deletion strain of the porcine pseudorabies virus by using the autonomously designed primer and probe.

In order to accurately, normatively, quickly and efficiently carry out the differential diagnosis of the pseudorabies virus wild virus and the gI/gE gene deletion vaccine strain in a pig farm, the invention provides a method for distinguishing the pseudorabies virus wild virus pathogen and the gI/gE gene deletion vaccine strain based on real-time fluorescent quantitative PCR (qPCR) of TaqMan.

In order to realize the purpose of the invention, in a first aspect, the invention provides a primer and a probe for identifying a gI/gE gene deletion vaccine strain and a wild strain of porcine pseudorabies virus by fluorescent quantitative PCR (polymerase chain reaction), wherein the nucleotide sequence of the primer for identifying gI/gE gene deletion is shown as SEQ ID NO. 1-2; the nucleotide sequence of the TaqMan probe for identifying the gI/gE gene deletion is shown in SEQ ID NO. 3.

Specifically, the primer and the probe provided by the invention are mainly suitable for identifying porcine pseudorabies virus gI/gE gene deletion vaccine strains HB98 and HB2000, and a fragment obtained by amplification of the primer provided by the invention is positioned at the junction of gI and gE genes.

In a second aspect, the invention provides a kit for identifying a gI/gE gene deletion vaccine strain and a wild strain of porcine pseudorabies virus by fluorescent quantitative PCR detection, which comprises the primer and the probe.

The kit provided by the invention also comprises a negative control and a positive control, wherein the positive control is nucleic acid which is a PRV positive sample detected by a laboratory, and the negative control is nucleic acid which is a PRV negative sample detected by the laboratory.

The invention extracts nucleic acid from the positive and negative controls for the first time, thereby reasonably and effectively reducing the influence of background nucleic acid on the experimental result.

The kit provided by the invention also comprises a hot start DNA polymerase modified based on a chemical method, a dUTP/UNG anti-pollution system and an anti-PCR inhibitor factor. In the kit provided by the invention, the used enzyme is chemically modified hot-start DNA polymerase, and is matched with the optimal Buffer optimized for qPCR, so that nonspecific amplification can be effectively inhibited, and high-sensitivity qPCR reaction can be carried out. Meanwhile, a dUTP/UNG anti-pollution system is introduced, so that the pollution from the previous amplification product can be completely eliminated, and the anti-PCR inhibitor factor is added into the reaction liquid, so that the method provided by the invention is more suitable for the detection of complex animal clinical samples.

In a third aspect, the invention provides a method for identifying a porcine pseudorabies virus vaccine strain and a wild strain for non-disease diagnosis, wherein the primer and the probe or the kit are used for carrying out qPCR amplification reaction on a sample to be detected.

In the method provided by the invention, the sample to be detected is the DNA of the porcine pseudorabies virus in samples such as porcine serum, excrement, semen, tissues, cell cultures and the like.

In the method provided by the invention, the qPCR reaction system is 2 XPrimer-one step-mix, 10 mul; f-primer, 0.5. mu.l; r-primer, 0.5. mu.l; probe, 1 μ l; RNase-Free-H₂O, 6 μ l; DNA, 2. mu.l; the qPCR reaction procedure was: at 95 ℃ for 2 min; 95 ℃ for 10 s; 60 ℃ for 35 s; 40 cycles.

In the method provided by the invention, the result judgment standard of the qPCR amplification reaction is as follows:

(1) the Ct value of the sample is less than or equal to 35, and the sample is judged to be positive;

(2) if the Ct value of the sample is more than 35 and less than or equal to 38, if the logarithmic amplification curve of the amplification curve is adopted, the sample is judged to be a suspicious positive sample, otherwise, the sample is judged to be negative;

rechecking the suspicious positive sample, if the Ct value of the rechecked sample is less than or equal to 35, judging the suspicious positive sample as positive, otherwise, judging the sample as negative;

(3) the sample has no Ct value or the Ct value is more than 38, and the sample is judged to be negative.

According to the understanding of the skilled person, the invention also claims the use of the above primers and probes or the above kit or the above method in epidemiological investigation of porcine pseudorabies virus. And the application of the primer and the probe or the kit or the method in the quality detection of the porcine pseudorabies virus vaccine.

The invention has the beneficial effects that:

(1) the primer and the probe provided by the invention have high sensitivity, the lowest detection limit is 189copies/uL, and all porcine pseudorabies virus gI/gE gene deletion vaccine strains and wild strains in the market can be distinguished;

(2) the kit provided by the invention is suitable for detecting complex animal clinical samples, such as porcine pseudorabies virus in samples of porcine serum, excrement, semen, tissues, cell cultures and the like;

(3) the identification method provided by the invention can accurately and efficiently identify the porcine pseudorabies virus gI/gE gene deletion vaccine strains HB98 and HB 2000.

Drawings

FIG. 1 is a standard curve chart of example 2 of the present invention.

FIG. 2 is a graph showing the sensitivity test in example 2 of the present invention.

FIG. 3 is a FAM channel result diagram of the positive and negative comparative amplification of primers PrV-gI/gE and PrV-gI/gE-C of the present invention.

FIG. 4 is a result chart of FAM and HEX channels amplified by national standard yin-yang contrast.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

Example 1

In this example, the junction of gI and gE was selected as the target region of the primer, and the amplification primer and TaqMan probe were designed for specific detection. The nucleotide sequence of the target region selected by the primer designed in the embodiment is shown in SEQ ID NO. 4.

This example performed nucleic acid extraction on negative and positive controls, which reduced the effect of background nucleic acid on the results of the experiment. The enzyme used in the embodiment is chemically modified hot-start DNA polymerase, and can effectively inhibit non-specific amplification and perform high-sensitivity qPCR reaction by matching with the optimal Buffer optimized for qPCR. And meanwhile, a dUTP/UNG anti-pollution system is introduced, so that the influence of pollution from a previous amplification product on qPCR can be completely eliminated. anti-PCR inhibitor factors are added into the reaction solution, so that the method provided by the embodiment is more suitable for detecting complex animal clinical samples.

The real-time fluorescent quantitative PCR method is adopted to detect the DNA of the porcine pseudorabies virus in samples such as porcine serum, feces, semen, tissues, cell cultures and the like, and is suitable for differential diagnosis of the porcine pseudorabies virus wild virus and gI/gE gene deletion vaccine and epidemiological investigation of PRV.

The primers and TaqMan probes for identifying the wild strain of porcine pseudorabies and the gI/gE gene deletion vaccine provided in the example are shown in Table 1.

TABLE 1 primers and TaqMan probes for real-time fluorescent quantitative PCR detection

The reaction system and the reaction program for real-time fluorescent quantitative PCR detection of the wild strain of the porcine pseudorabies virus and the gI/gE gene deletion vaccine strain provided by the embodiment are as follows:

the reaction system is as follows: 2 × primer-one step-mix, 10 μ l; f-primer, 0.5. mu.l; r-primer, 0.5. mu.l; probe, 1 μ l; RNase-Free-H₂O，6μl；DNA，2μl。

The reaction procedure is as follows: at 95 ℃ for 2 min; 95 ℃ for 10 s; 60 ℃ for 35 s; 40 cycles.

Example 2

1. Establishment of a standard curve: the recombinant plasmid carrying gI and gE genes with correct sequencing is subjected to Nanodrop concentration determination, the copy number of each standard substance is calculated, and the positive standard substances are respectively diluted to 4 multiplied by 10 according to a 10-fold gradient¹⁰、4×10⁹、4×10⁸、4×10⁷、4×10⁶、4×10⁵、4×10⁴、4×10³、4×10²、4×10¹、4×10⁰copies/. mu.L, each sample is repeated for 2 times, and standard substances with different concentrations are taken as templates to perform real-time fluorescent quantitative PCR detection under the guidance of primers and TaqMan probes.

After the detection is finished, the concentration Log value (X axis) of each standard substance is plotted against the corresponding Ct value (Y axis) of the standard substance to draw a standard curve, the standard curve is shown as figure 1, and the correlation coefficient is R²When the error is smaller than 0.997, a standard curve is available and obtained by the standard curveThe linear equation to is: y-0.997 x + 52.781. The result judgment standard is as follows: the Ct value is less than or equal to 35, the Ct value is more than 35 and less than or equal to 38, the Ct value is suspicious, and the Ct value is more than 38, the Ct value is negative.

2. Sensitivity test: the concentration of the positive standard substance is measured by a spectrophotometer and converted into copies/uL, 10-fold gradient dilution is carried out, the sample is used as a template for qPCR amplification, the test result is shown in figure 2, and the lowest detection limit of the positive control standard substance (FAM channel) is 189copies/uL, which shows that the method has high sensitivity.

3. Specific experiments: a commercial kit (a column extraction method or a magnetic bead method) is used for respectively carrying out nucleic acid extraction on negative reference samples of Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV2), Porcine Parvovirus (PPV), porcine encephalitis B virus (JEV), Mycoplasma Hyopneumoniae (MH), Porcine Epidemic Diarrhea Virus (PEDV) and PK-15 cells, and a blank system control and a negative-positive control are set at the same time, wherein the specific samples are as follows: CSFV +, 2 PRRSV +, 3 PCV2+, 4 PRV +, 5 PPV +, 6 JEV +, 7 MH +, 8 PEDV +, 9 PK-15 cells, 10 blank system control, 11 negative control, 12 positive control.

All pathogen samples are derived from daily detection positive samples in the laboratory, are verified to be specific pathogen positive nucleic acids through PCR, and the other samples are verified to be PRV negative through PCR except the No.4 PRV positive sample, the primers obtained through the design of the embodiment 1 and national standard primers are used for detecting the samples, and the detection results of the two samples are compared.

The quality control result of the primer designed in the embodiment 1 is shown in fig. 3, and the FAM channel of the negative reference sample of the primer and probe set has no typical S-shaped curve and no Ct value; the Ct value of the positive sample is less than or equal to 30, which indicates that the negative and positive controls are established, the quality control extraction process is normal, the detection result of the sample is shown in Table 2, the Ct value of the No.4 sample detected in the embodiment is less than or equal to 35, which is a typical S-shaped amplification curve, and the report is positive;

the quality control result of the national standard primer is shown in FIG. 4, the PRV negative control FAM channel does not report a Ct value and has no typical S-type amplification curve, and the HEX channel does not report a Ct value and has no typical S-type amplification curve; the Ct value of the positive control FAM channel is less than or equal to 30, the Ct value of the HEX channel is less than or equal to 30, the amplification curves of the two channels are both typical S-shaped, and the positive and negative control is established; according to national standard: the Ct value of the FAM channel of the sample to be detected is less than or equal to 38, the Ct value of the HEX channel is less than or equal to 38, the amplification curves are typical S types, and the result is reported as PRV nucleic acid positive; the Ct value of the FAM channel of the sample to be detected is less than or equal to 40, the amplification curve is a typical S type, the HEX channel has no Ct value or no typical S amplification curve, and the result is reported as the PRVgE deleted strain nucleic acid positive; and in the detection result of the detected sample, both the FAM channel and the HEX channel have no Ct value or a typical S amplification curve, and the result is reported as PRV nucleic acid negative.

TABLE 2 results of specificity experiments

The primers used by FAM channels of the national standard primers are used for amplifying wild virus genes so as to judge whether PRV nucleic acid is contained, and the primers used by HEX channels are used for amplifying gE genes so as to judge whether the gE genes are deleted. The detection results of the samples are shown in table 2, and the No.4 sample report in the national standard primer detection is judged to be PRV nucleic acid positive and is a non-PRVgE deletion strain.

The test results in Table 2 show that the primer amplification detection results in example 1 are consistent with the results obtained by national standard primer detection. It is shown that the primer and probe provided in example 1 of the present invention have high specificity, i.e., the identification method provided by the present invention has good specificity.

4. Clinical sample alignment experiment: 6 parts (3 parts positive and 3 parts negative) of clinical samples with clear background, 1 part of each of the Korea HB98 vaccine and the HB2000 vaccine are detected by using the kit, the total amount of the samples is 8, and the test sample adding sequence number is as follows: PRV +, 2.PRV +, 3.PRV +, 4.PRV-, 5.PRV-, 6.PRV-, 7.HB98, 8.HB2000, 9. blank negative control, 10. negative control, 11. positive control. 2ul of template was taken and 2 replicates of each pair of primers were made, the results are shown in Table 3.

TABLE 3 clinical sample experiments

The results in Table 3 show that the test results of the PRV-gI/gE group are consistent with those of the national standard group, the Ct value of the FAM channel in the detection results of 3 positive samples is less than or equal to 35, the amplification curve is a typical S-shaped amplification curve and is reported as PRV nucleic acid positive, HB98 and HB2000 vaccine samples in the PRV-gI/gE primer test are negative, and the detection results of the national standard group are reported as HB98 and HB2000 vaccine samples are PRV positive.

5. Comparison between kits experiment: selecting a PRVgE nucleic acid qPCR detection kit for replacing Tokyo province and Weidi detection in a meter factory, performing a comparative test with the method, and using 6 parts (3 parts positive and 3 parts negative) of clinical samples with clear backgrounds, 1 part of each of a Kogya HB98 vaccine and an HB2000 vaccine, and 8 parts of samples in total, wherein the test sample adding sequence number is as follows: the experiment was carried out using 1.PRV +, 2.PRV +, 3.PRV +, 4.PRV-, 5.PRV-, 6.PRV-, 7.HB98, 8.HB2000, 9. blank negative control, 10. negative control, 11. positive control. 2ul of template was taken and 2 replicates of each pair of primers were made, the results are shown in Table 4.

TABLE 4 results of the comparison between the kits

The results in Table 4 show that the results of the positive samples of the PrV-gI/gE group tests are consistent with the results of the Henren Henry and Weidi detection, the FAM channel Ct value in the detection results of 3 positive samples is less than or equal to 35, the amplification curve is a typical S-shaped amplification curve, and the result is reported as PRV nucleic acid positive.

HB98 and HB2000 vaccine samples in the test of the PrV-gI/gE group are PRV negative, and the test of century Yuan Heng and Meerweidi is PRV positive.

Example 3 identification of commercial porcine pseudorabies virus gI/gE Gene-deleted vaccine strains and wild strains

In this example, the porcine pseudorabies virus gI/gE gene-deleted vaccine strains of the Korea HB98, the Korea HB2000, the Mediterranean HB2000, the Yibang K61, the Kazakh K61, and the Boringer Vargham K61 strain were tested using the same primers and probes as in example 1 and the same qPCR amplification system and reaction procedure as in example 1. The results are shown in Table 5, the PrV-gI/gE primers identify that the 6 vaccines are negative, which indicates that the 6 vaccines are all gene-deleted vaccine strains, and national standard primers can only identify the Yibang K61, Harbin K61 and Boringer Invitrogen K61 strains as gene-deleted vaccine strains.

TABLE 5 detection results of vaccines from different manufacturers

Comparative example 1

The comparative example is the same as the example 1, the joint of gI and gE is selected as a target region for primer design, and an amplification primer and a TaqMan probe are designed for specific detection. The primers and probes thus designed are shown in Table 6.

TABLE 6 primers and TaqMan probes for real-time fluorescent quantitative PCR detection of comparative examples

The quality control results of the probe and the primer designed by the comparative example are shown in FIG. 3, and 8 samples (3 positive samples, 3 negative samples, a Kokai HB98 vaccine strain and an HB2000 vaccine strain) which are the same as those in example 2 are detected by using the primer and the probe designed by the comparative example. The results are shown in Table 7.

TABLE 7 comparative example primers and Probe test results

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims (10)

1. The primers and the probes for detecting and identifying the gI/gE gene deletion vaccine strain and the wild strain of the porcine pseudorabies virus by fluorescent quantitative PCR are characterized in that the nucleotide sequence of the primers is shown as SEQ ID NO. 1-2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3.

2. The primers and probes as claimed in claim 1, wherein the gI/gE gene-deleted vaccine strain of porcine pseudorabies virus is HB98 and HB 2000.

3. A kit for detecting and identifying gI/gE gene deletion vaccine strains and wild strains of porcine pseudorabies viruses by fluorescent quantitative PCR (polymerase chain reaction), which is characterized by comprising the primer and the probe of claim 1.

4. The kit of claim 3, further comprising a negative control and a positive control, wherein the positive control is a nucleic acid that is a PRV positive sample detected by a laboratory, and the negative control is a nucleic acid that is a PRV negative sample detected by the laboratory.

5. The kit of claim 4, further comprising a chemically modified hot-start DNA polymerase, dUTP/UNG antipollution system, anti-PCR inhibitor factor.

6. A method for identifying a porcine pseudorabies virus vaccine strain and a wild strain for non-disease diagnosis, which is characterized in that a qPCR amplification reaction is carried out on a sample to be tested by using the primer and the probe of claim 1 or the kit of any one of claims 3 to 5.

7. The method according to claim 6, wherein the sample to be tested is porcine pseudorabies virus DNA in porcine serum, feces, semen, tissue, cell culture and other samples.

8. The method of claim 7, wherein the qPCR reaction system is 2 XPrimer-one step-mix, 10 μ l; f-primer, 0.5. mu.l; r-primer, 0.5. mu.l; probe, 1 μ l; RNase-Free-H₂O, 6 μ l; DNA, 2. mu.l; the qPCR reaction procedure was: at 95 ℃ for 2 min; 95 ℃ for 10 s; 60 ℃ for 35 s; 40 cycles.

9. The method of claim 8, wherein the result determination criteria for the qPCR amplification reaction are:

(1) the Ct value of the sample is less than or equal to 35, and the sample is judged to be positive;

(2) if the Ct value of the sample is more than 35 and less than or equal to 38, if the logarithmic amplification curve of the amplification curve is adopted, the sample is judged to be a suspicious positive sample, otherwise, the sample is judged to be negative;

rechecking the suspicious positive sample, if the Ct value of the rechecked sample is less than or equal to 35, judging the suspicious positive sample as positive, otherwise, judging the sample as negative;

(3) the sample has no Ct value or the Ct value is more than 38, and the sample is judged to be negative.

10. Use of the primers and probes of any of claims 1-2, or the kit of any of claims 3-5, or the method of any of claims 6-9 for epidemiological investigation of porcine pseudorabies virus.

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