CN110724770A - Anti-pollution fluorescent quantitative PCR (polymerase chain reaction) primer group, kit and detection method for detecting African swine fever virus - Google Patents
Anti-pollution fluorescent quantitative PCR (polymerase chain reaction) primer group, kit and detection method for detecting African swine fever virus Download PDFInfo
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Abstract
The invention discloses an anti-pollution fluorescent quantitative PCR primer group, a kit and a detection method for detecting African swine fever virus. The primer comprises a primer and a probe, wherein the nucleotide sequence of the primer is shown as SEQ ID NO. 1-3; the kit comprises the primer, the probe and the reaction reagent. When the kit is used, an S-type amplification curve appears in an FAM channel and is an African swine fever virus infection, and a non-S-type amplification curve is a negative African swine fever virus infection. The kit provided by the invention can effectively prevent aerosol pollution, is simple to operate, low in cost, high in sensitivity and good in specificity, and is suitable for export quarantine, food sanitation and African swine fever virus detection of livestock farms.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fluorescent quantitative PCR primer, a kit and a detection method for detecting African swine fever virus.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease caused by African Swine Fever Virus (ASFV) infection. The world animal health Organization (OIE) ranks the animal epidemic disease as a legally reported animal epidemic disease, and China ranks the animal epidemic disease as a type of animal epidemic disease. The African swine fever is not pathogenic to human and livestock, but the incidence and the fatality rate of the swine after infection can reach 100 percent.
At present, the laboratory detection method of the ASF mainly comprises virus separation, indirect immunofluorescence assay, ELISA, PCR and the like. Although virus separation and indirect immunofluorescence tests belong to standard detection methods, the operation is complicated, and the tests are difficult to be carried out in laboratories with common conditions. The fluorescent quantitative PCR technology has the advantages of simple operation, high sensitivity, good specificity and the like, and is applied to the detection of the African swine fever virus. However, the fluorescence quantitative PCR has the disadvantage of being easily contaminated by aerosol, and even a trace amount of amplification product leaks out, false positive may be generated. A fluorescent quantitative PCR detection method for African swine fever viruses is established by targeting P72 in the patents of molecular probes for detecting African swine fever viruses, kits and applications thereof (application number: 201910088473.4, application date: 2019-01-30; publication number: 109781981, publication date: 2019-05-21), fluorescent PCR rapid detection kits for African swine fever viruses (application number: 201910108734.4, application date: 2019-02-03; publication number: 109593893, publication date: 2019-04-09), and fluorescent PCR detection reagents and detection methods for African swine fever viruses (application number: 201811639635.0, application date: 2018-12-29; publication number: 109504806, publication date: 2019-03-22). In the current high-throughput sample detection process, the fluorescence quantitative detection method targeting the P72 gene is easy to generate false positive once aerosol pollution is generated. The research shows that the ASFV genome DNA is about 170Kb and encodes more than 200 proteins. The P72 gene is not the only conserved gene of ASFV, so the African swine fever virus fluorescent quantitative PCR detection method is established by targeting the E184L gene in the patent of African swine fever virus detection kit and detection method based on the E184L gene (application number: 201811202105.X, application date: 2018-10-16; publication number: 109266786, publication date: 2019-01-25), and the African swine fever virus fluorescent quantitative PCR detection method shows good specificity and sensitivity. However, this method is also not effective for improving the aerosol contamination problem.
Therefore, the development of an anti-pollution fluorescent quantitative PCR detection method for diagnosing African swine fever virus is imperative.
Disclosure of Invention
The invention aims to provide an anti-pollution fluorescent quantitative PCR primer for detecting African swine fever virus, provide an anti-pollution fluorescent quantitative PCR rapid detection kit and provide a detection method of the kit.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
an anti-pollution fluorescent quantitative PCR primer group for detecting African swine fever virus is characterized in that the primer group comprises an upstream primer DP-P1, a downstream primer DP-P2 and a probe DP-Pb of a targeted DNA primase gene;
the nucleotide sequence of the primer DP-P1 is as follows: 5'-ACGCATTCTTCAGCATACTCG-3', respectively;
the nucleotide sequence of the primer DP-P2 is as follows: 5'-CCTCCGTTTCGCTCTCAAT-3', respectively;
the nucleotide sequence of the probe DP-Pb is as follows: 5'-TACAGAAGGAATACAACGGGCA-3' are provided.
Further, the probe DP-Pb was labeled with 6FAM at the 5 'end and BHQ1 at the 3' end.
An anti-pollution fluorescent quantitative PCR kit for detecting African swine fever virus comprises the primer group mixed solution, wherein the final concentration of the primer DP-P1 is 0.2 mmol/L; the final concentration of the primer DP-P2 is 0.2 mmol/L; the final concentration of the probe DP-Pb is 0.2 mmol/L.
Further, the kit also comprises an anti-pollution fluorescent quantitative PCR premix solution, wherein the anti-pollution fluorescent quantitative PCR premix solution contains dUTP and UNG enzyme (Uracil-N-Glycosylase). If dUTP is used for replacing dTTP in the reaction system, the PCR product is DNA double-chain containing dUTP, a pretreatment step of 37 ℃ is added before the PCR reaction is started, and UNG enzyme can eliminate dUTP-DNA double-chain pollutants possibly existing in the reaction system by degrading dUTP. After the denaturation step is started, the UNG enzyme is inactivated, and the normal running of the PCR reaction is not influenced.
Furthermore, the kit also comprises a positive control, wherein the positive control is a recombinant plasmid carrying a DNA primase gene.
The primer group or the kit is applied to the detection of African swine fever virus.
The detection method for detecting the African swine fever virus by using the kit comprises the following steps:
1) preparing a primer mixed solution by using the primer, wherein the final concentration of the primer DP-P1 is 0.2 mmol/L; the final concentration of the primer DP-P2 is 0.2 mmol/L; the final concentration of the probe DP-Pb is 0.2 mmol/L;
2) carrying out reaction detection on a sample to be detected by using the primer mixed solution and the anti-pollution fluorescent quantitative PCR premixed solution; the reaction system is 20 mu L, wherein the primer mixed solution is 2 mu L, the fluorescent quantitative PCR premixed solution is 10 mu L, the sample DNA to be detected is 2 mu L, and the DNaseffee water is 6 mu L;
3) and (4) interpretation of results:
a) negative: if the S-type amplification curve does not exist, the fact that the sample to be detected is not infected by the African swine fever virus is proved;
b) positive: if an S-type amplification curve appears in the FAM channel, the sample to be detected is proved to be infected by the African swine fever virus.
Further, the reaction conditions in step 2) include: incubating at 37 ℃ for 2 min; pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 10 s; 60 ℃, 30s, 45 cycles.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1) the kit provided by the invention can prevent aerosol pollution, dUTP replaces dTTP in a reaction system, and dUTP-DNA double-chain pollutants possibly existing in the reaction system are eliminated.
2) The kit provided by the invention has good specificity, and shows negative reaction to the genomic DNA of serum type 5 haemophilus parasuis, serum type 2 streptococcus suis, swine erysipelas bacillus, swine infectious actinobacillus pleuropneumoniae, porcine pseudorabies virus and porcine circovirus type 2; the sensitivity is high, and 10 copies of African swine fever virus DNA primase genes can be detected at the lowest.
3) The detection kit provided by the invention is simple to operate, low in cost and very suitable for African swine fever virus detection in export quarantine, food sanitation and livestock farms.
The kit provided by the invention can prevent aerosol pollution, has strong specificity and high sensitivity, and can meet the requirements of high-throughput and accurate and rapid detection of ASFV. The invention ensures that the detection process of the African swine fever virus is more convenient, rapid and accurate.
Drawings
FIG. 1 is a diagram showing the results of the fluorescent quantitative PCR kit method of the present invention.
FIG. 2 is a diagram showing the results of the specific experiments of the fluorescent quantitative PCR kit provided by the present invention.
FIG. 3 is a diagram showing the results of the sensitivity test of the fluorescent quantitative PCR kit provided by the present invention.
Detailed Description
The following detailed description will be provided to explain embodiments of the present invention in conjunction with the accompanying drawings, so as to fully understand and implement how to implement the technical means for solving the technical problems and achieving the technical effects of the present invention.
Materials referred to in the examples:
a) the African swine fever virus DNA primase gene is synthesized by Shanghai biological engineering technical service company Limited; the primers and the probes are synthesized by Shanghai biological engineering technology service company Limited; the anti-pollution fluorescent quantitative PCR premix is purchased from Nanjing NuoZan Biotech Co., Ltd (Q113-02); viral nucleic acid extraction kits were purchased from magenta (meiy) organisms (R4410-03); pseudorabies live vaccine (Bartha-K61 strain) and porcine circovirus type 2 inactivated vaccine (LG strain) were purchased from Shanghai Haili Biotechnology Ltd;
b) the following biological materials are disclosed in the literature "establishment and application of dual PCR method for Haemophilus parasuis and Streptococcus suis. Chinese veterinary science, 2009(9): 1155 and 1157.": haemophilus parasuis serotype 5, streptococcus suis serotype 2, actinobacillus pleuropneumoniae.
Example 1 design and Synthesis of PCR primer set
According to the Primer design principle, aiming at the conserved region sequence of the African swine fever virus DNA primase gene, the Primer 5 software is used for designing a PCR Primer and a probe, and the GC content of the Primer is ensured to be between 40 and 60 percent according to our experience. After screening of a plurality of sets of primers and probe combinations, the primer sequences of the invention are finally obtained as shown in Table 1, the primers are synthesized by Shanghai biological engineering technical service Co., Ltd, and the synthesized primers are diluted into 10 mu M with sterilized triple distilled water and stored at-20 ℃.
TABLE 1 primer set for fluorescent quantitative PCR
Example 2 application of fluorescent quantitative PCR primers in detection of viruses to be detected
Firstly, preparation of nucleic acid standard substance
Performing nucleic acid on African swine fever virus DNA primase gene synthesized by Shanghai Biotechnology engineering service Co., LtdAfter the concentration measurement, the mixture was diluted with ultrapure water to a concentration of 0.4 ng/. mu.L (1.2X 10)5Copy), as a nucleic acid standard.
Second, establishment of fluorescent quantitative PCR detection method
The concentration was taken to be 0.4 ng/. mu.L (1.2X 10)5Copy) of African swine fever virus DNA primase gene DNA as a template, and applying primers and probes obtained by screening (shown in table 1), preparing a reaction system according to table 2, wherein the reaction condition is incubation for 2min at 37 ℃; pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 10 s; 60 ℃, 30s, 45 cycles. The results obtained are shown in FIG. 1: 1 is blank control, and 2 is African swine fever virus DNA primase gene DNA. The results show that: the blank control does not have an S-type amplification curve, and an S-type amplification curve is formed in a FAM channel by taking the DNA of the DNA primase gene of the African swine fever virus as a template, which indicates that the kit provided by the invention can quickly and accurately detect the African swine fever virus.
TABLE 2 fluorescent quantitative PCR reaction System
Third, specificity of fluorescent quantitative detection method
The specificity of the detection system is detected by using the genome DNA of porcine pseudorabies virus, porcine circovirus type 2, erysipelothrix rhusiopathiae, porcine infectious actinobacillus pleuropneumoniae, serum type 5 haemophilus parasuis and serum type 2 streptococcus suis as a template. The reaction system is shown in Table 2, and the reaction condition is incubation for 2min at 37 ℃; pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 10 s; 60 ℃, 30s, 45 cycles. The results obtained are shown in FIG. 2: wherein: 1 is a detection result by taking a serum 5 type haemophilus parasuis genome DNA as a template; 2, taking the genome DNA of the streptococcus suis serotype 2 as a detection result of a template; 3, taking erysipelothrix rhusiopathiae genome DNA as a detection result of a template; 4, taking the genome DNA of the actinobacillus pleuropneumoniae as a detection result of a template; 5, taking the genome DNA of the porcine pseudorabies virus as a detection result of a template; 6, taking the porcine circovirus type 2 genome DNA as a detection result of a template; 7 is African swine fever virus DNA primase gene DNA (1.2X 10)3Copy) as a moduleThe detection result of the plate; 8 is a blank control; the serous Haemophilus parasuis type 5, serous Streptococcus suis type 2, erysipelothrix rhusiopathiae, Actinobacillus pleuropneumoniae, pseudorabies virus and porcine circovirus type 2 are taken as templates, respectively, and an S-type amplification curve does not appear in the FAM channel, and the result shows that the specificity of the detection system is good, and the African swine fever virus can be specifically identified and detected.
Fourth, sensitivity of fluorescent quantitative PCR detection method
The concentration of 10. mu.L was taken to be 0.4 ng/. mu.L (1.2X 10)5Copy) of African swine fever virus DNA primase gene DNA, adding 80 μ L of sterilized pure water for 10-fold dilution, taking 10 μ L of the 10-fold dilution, sequentially adding 90 μ L of sterilized pure water for 10-fold gradient dilution, taking 2 μ L of each diluted sample solution for fluorescent quantitative PCR detection (the reaction system is shown in Table 2; the reaction conditions were all incubation at 37 ℃ for 2min, except for changes in template; pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 10 s; 60 ℃, 30s, 45 cycles). The results obtained are shown in FIG. 3: 1 is 1.2X 105Copy of African swine fever virus DNA primase gene; 2 is 1.2X 104Copy of African swine fever virus DNA primase gene; 3 is 1.2X 103Copy of African swine fever virus DNA primase gene; 4 is 120 copies of African swine fever virus DNA primase gene; 5 is 12 copies of African swine fever virus DNAprimese gene; 6 is 1.2 copies of African swine fever virus DNA primase gene; 7 is 0.12 copies of African swine fever virus DNA primase gene; blank control 8. An S-type amplification curve appears on a FAM channel by taking the African swine fever virus DNA primase gene DNA diluted by No. 1-5 as a template, and the S-type amplification curve does not appear by taking the African swine fever virus DNA primase gene DNA diluted by No. 6-8 as the template, and the result shows that the method can detect 12 copies of the African swine fever virus DNAprimase gene DNA at the lowest.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (10)
1. A fluorescence quantitative PCR primer group for detecting African swine fever virus comprises an upstream primer DP-P1, a downstream primer DP-P2 and a probe DP-Pb of a targeted DNA primase gene;
the nucleotide sequence of the primer DP-P1 is as follows: 5'-ACGCATTCTTCAGCATACTCG-3', respectively;
the nucleotide sequence of the primer DP-P2 is as follows: 5'-CCTCCGTTTCGCTCTCAAT-3', respectively;
the nucleotide sequence of the probe DP-Pb is as follows: 5'-TACAGAAGGAATACAACGGGCA-3' are provided.
2. The fluorogenic quantitative PCR primer of claim 1, wherein the probe DP-Pb is labeled with 6FAM at the 5 'end and BHQ1 at the 3' end.
3. An anti-contamination fluorescent quantitative PCR kit for detecting African swine fever virus, comprising the primer set of claim 1 or 2.
4. The PCR kit of claim 3, further comprising a pre-mix of anti-contamination fluorescent quantitative PCR.
5. The PCR kit of claim 3, wherein the pre-contamination-resistant fluorescent quantitative PCR mix contains dUTP and UNG enzyme.
6. The PCR kit of claim 3, wherein the kit further comprises a positive control.
7. The test kit according to claim 6, wherein the positive control is a recombinant plasmid carrying a DNA primase gene.
8. Use of the primer set according to claim 1 or the kit according to claim 3 for the detection of African swine fever virus.
9. A fluorescence quantitative PCR detection method for detecting African swine fever virus is characterized by comprising the following steps:
1) the primer mixture was prepared using the primer set of claim 1, wherein the final concentration of the primer DP-P1 was 0.2 mmol/L; the final concentration of the primer DP-P2 is 0.2 mmol/L; the final concentration of the probe DP-Pb is 0.2 mmol/L;
2) detecting a sample to be detected by using a primer mixed solution and an anti-pollution fluorescent quantitative PCR premixed solution; the reaction system is 20 mu L, wherein the primer mixed solution is 2 mu L, the fluorescent quantitative PCR premixed solution is 10 mu L, the sample DNA to be detected is 2 mu L, and the DNase free water is 6 mu L;
3) and (4) interpretation of results:
a) negative: if the S-type amplification curve does not exist, the fact that the sample to be detected is not infected by the African swine fever virus is proved;
b) positive: if an S-type amplification curve appears in the FAM channel, the sample to be detected is proved to be infected by the African swine fever virus.
10. The detection method according to claim 6, wherein the reaction conditions in step 2) include: incubating at 37 ℃ for 2 min; pre-denaturation at 95 ℃ for 5 min; 95 ℃ for 10 s; 60 ℃, 30s, 45 cycles.
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| EP1033405A2 (en) * | 1999-02-25 | 2000-09-06 | Ceres Incorporated | Sequence-determined DNA fragments and corresponding polypeptides encoded thereby |
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