CN110724770A - Anti-pollution fluorescent quantitative PCR primer set, kit and detection method for detection of African swine fever virus - Google Patents

Abstract

The invention discloses an anti-pollution fluorescent quantitative PCR primer set, a kit and a detection method for detecting African swine fever virus. The primers include primers and probes whose nucleotide sequences are shown in SEQ ID NOs. 1 to 3; the kit includes the aforementioned primers, probes, and reaction reagents. When the kit is used, an S-type amplification curve appears in the FAM channel, which means African swine fever virus infection, and no S-type amplification curve is negative for African swine fever virus infection. The kit provided by the invention has the advantages of effectively preventing aerosol pollution, simple operation, low cost, high sensitivity and good specificity, and is suitable for export quarantine, food hygiene and African swine fever virus detection in animal husbandry farms.

Description

Anti-pollution fluorescent quantitative PCR primer sets, kits and assays for the detection of African swine fever virus test method

technical field

The invention belongs to the field of biotechnology, and in particular relates to a fluorescent quantitative PCR primer, a kit and a detection method for detecting African swine fever virus.

Background technique

African swine fever (ASF) is an acute, febrile and highly contagious infectious disease caused by African swine fever virus (ASFV). The World Organization for Animal Health (OIE) lists it as a statutory notifiable animal disease, and China lists it as a Class I animal disease. African swine fever is not pathogenic to humans and is not a zoonotic disease, but the morbidity and mortality of pigs can be as high as 100%.

At present, the laboratory detection methods of ASF mainly include virus isolation, indirect immunofluorescence test, ELISA, PCR and so on. Although virus isolation and indirect immunofluorescence test are standard detection methods, they are cumbersome and difficult to carry out in laboratories with ordinary conditions. Fluorescence quantitative PCR technology has the advantages of simple operation, high sensitivity and good specificity, and has been applied in the detection of African swine fever virus. However, real-time PCR also has the disadvantage of being easily contaminated by aerosols, and even if a small amount of amplification product leaks, it will lead to false positives. Patent "Molecular Probes, Kits and Applications for the Detection of African Swine Fever Virus" (application number: 201910088473.4, application date: 2019-01-30; publication number: 109781981, publication date: 2019-05-21), patent " African Swine Fever Virus Fluorescent PCR Rapid Detection Kit (Application No.: 201910108734.4, Application Date: 2019-02-03; Publication No.: 109593893, Publication Date: 2019-04-09), patent "A kind of African swine fever fluorescent PCR Detection Reagents and Detection Methods" (application number: 201811639635.0, application date: 2018-12-29; publication number: 109504806, publication date: 2019-03-22), etc., have established African swine fever virus fluorescence quantitative PCR targeting P72 Detection method. In the current high-throughput sample detection process, once aerosol pollution occurs, these fluorescent quantitative detection methods targeting the P72 gene are prone to false positives. Studies have shown that ASFV genomic DNA is about 170Kb, encoding more than 200 proteins. The P72 gene is not the only conserved gene of ASFV, so the patent "African swine fever virus detection kit and detection method based on E184L gene" (application number: 201811202105.X, application date: 2018-10-16; publication number: 109266786, published Date: 2019-01-25) A quantitative PCR detection method for African swine fever virus was established targeting the E184L gene, showing good specificity and sensitivity. However, this method has not been effectively improved against the problem of aerosol pollution.

Therefore, it is imperative to develop an anti-pollution fluorescent quantitative PCR detection method that can be used for the diagnosis of African swine fever virus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to firstly provide an anti-pollution fluorescent quantitative PCR primer for detecting African swine fever virus, secondly to provide the anti-pollution fluorescent quantitative PCR rapid detection kit, and thirdly to provide a detection method of the kit.

In order to achieve the above object, the concrete technical scheme that the present invention takes is:

An anti-pollution fluorescent quantitative PCR primer set for detecting African swine fever virus, characterized in that the primer set comprises an upstream primer DP-P1, a downstream primer DP-P2 and a probe DP-Pb targeting DNA primase gene;

The nucleotide sequence of the primer DP-P1 is: 5'-ACGCATTCTTCAGCATACTCG-3';

The nucleotide sequence of the primer DP-P2 is: 5'-CCTCCGTTTCGCTCTCAAT-3';

The nucleotide sequence of the probe DP-Pb is: 5'-TACAGAAGGAATACAACGGGCA-3'.

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, comprising the above-mentioned primer set mixture, wherein the final concentration of the primer DP-P1 is 0.2mmol/L; the final concentration of the primer DP-P2 is 0.2mmol/L /L; the final concentration of the probe DP-Pb was 0.2 mmol/L.

Further, the kit also includes an anti-pollution fluorescent quantitative PCR premix, and the anti-pollution fluorescent quantitative PCR premix contains dUTP and UNG enzymes (Uracil-N-Glycosylase). If dUTP is used to replace dTTP in the reaction system, the PCR product will be DNA double-strands containing dUTP, and a pretreatment step of 37°C is added before the PCR reaction starts. By degrading dUTP, UNG enzyme can degrade dUTP that may exist in the reaction system. Elimination of dUTP-DNA double-stranded contaminants. After the denaturation step begins, the UNG enzyme is inactivated and will no longer affect the normal progress of the PCR reaction.

Further, the kit also includes a positive control, and the positive control is a recombinant plasmid carrying a DNA primase gene.

Application of the above primer set or kit in detecting African swine fever virus.

A detection method for detecting African swine fever virus using the above-mentioned kit, comprising the following steps:

1) using the primers to configure the primer mixture, wherein the final concentration of primer DP-P1 is 0.2mmol/L; the final concentration of primer DP-P2 is 0.2mmol/L; the final concentration of probe DP-Pb is 0.2mmol/L;

2) Use the above-mentioned primer mixture and anti-pollution fluorescent quantitative PCR premix for reaction detection of the sample to be tested; the reaction system is 20 μL, wherein the primer mixture is 2 μL, the fluorescent quantitative PCR premix is 10 μL, the DNA of the sample to be tested is 2 μL, and the DNasefree water is 6 μL;

3) Interpretation of results:

a) Negative: If there is no S-type amplification curve, it proves that the sample to be tested has no African swine fever virus infection;

b) Positive: If an S-shaped amplification curve appears in the FAM channel, it proves that the sample to be tested is infected with African swine fever virus.

Further, the reaction conditions in step 2) include: incubation at 37°C for 2 min; pre-denaturation at 95°C for 5 min; 95°C, 10s; 60°C, 30s, 45 cycles.

Compared with the prior art, the advantages and beneficial effects of the present invention:

1) The kit provided by the present invention can prevent aerosol pollution, replace dTTP in the reaction system with dUTP, and eliminate the dUTP-DNA double-stranded pollutants that may exist in the reaction system.

2) The kit provided by the present invention has good specificity, and is suitable for serotype 5 Haemophilus parasuis, serotype 2 Streptococcus suis, Erysipelas suis, Actinobacillus pleuropneumoniae, porcine pseudorabies virus, porcine ringworm The genomic DNA of virus type 2 was negative; with high sensitivity, a minimum of 10 copies of the African swine fever virus DNA primase gene could be detected.

3) The detection kit provided by the present invention has simple operation and low cost, and is very suitable for export quarantine, food hygiene and African swine fever virus detection in animal husbandry farms.

The kit provided by the invention can prevent aerosol pollution, has strong specificity and high sensitivity, and is expected to meet the needs of high-throughput, accurate and rapid detection of ASFV. The invention makes the detection process of African swine fever virus more convenient, fast and accurate.

Description of drawings

FIG. 1 is a result diagram of the establishment of the fluorescence quantitative PCR kit method provided by the present invention.

FIG. 2 is a result diagram of the specific experiment of the fluorescence quantitative PCR kit provided by the present invention.

FIG. 3 is a graph showing the results of the sensitivity test of the fluorescence quantitative PCR kit provided by the present invention.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings, so as to fully understand and implement the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects.

Materials involved in the examples:

a) The African swine fever virus DNA primase gene was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.; the primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.; the anti-pollution fluorescent quantitative PCR premix was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Q113-02); viral nucleic acid extraction kit was purchased from Magen (Mecke) Bio (R4410-03); live pseudorabies vaccine (Bartha-K61 strain), porcine circovirus type 2 inactivated vaccine ( LG strain) was purchased from Shanghai Haili Biotechnology Co., Ltd.;

b) The following biological materials have been published in the document "Establishment and application of double PCR method for Haemophilus parasuis and Streptococcus suis. Chinese Veterinary Journal, 2009(9): pp. 1155-1157.": Serotype 5 parasuis Haemophilus suis, Streptococcus suis serotype 2, Actinobacillus pleuropneumoniae.

Example 1 Design and synthesis of PCR primer sets

According to the primer design principle, Primer 5 software was used to design PCR primers and probes for the conserved region sequence of the African swine fever virus DNA primase gene, and the GC content of the primers was guaranteed to be between 40% and 60% according to our experience. After multiple sets of primers and probe combinations are screened, the primer sequences of the present invention are finally obtained as shown in Table 1. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the synthesized primers were diluted with sterilized three-distilled water. The concentration was 10 μM and stored at -20°C.

Table 1 Primer sets for fluorescence quantitative PCR

Example 2 Application of fluorescent quantitative PCR primers in the detection of viruses to be tested

1. Preparation of nucleic acid standards

The nucleic acid concentration of the African swine fever virus DNA primase gene synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. was measured, and then diluted with ultrapure water to a concentration of 0.4 ng/μL (1.2×10 ⁵ copies) as a nucleic acid standard.

Second, the establishment of fluorescence quantitative PCR detection method

Take the African swine fever virus DNA primase gene DNA with a concentration of 0.4ng/μL (1.2×10 ⁵ copies) as the template, apply the primers and probes obtained by screening (as shown in Table 1), configure the reaction system according to Table 2, and react The conditions were incubation at 37°C for 2 min; pre-denaturation at 95°C for 5 min; 95°C, 10s; 60°C, 30s, 45 cycles. The obtained results are shown in Figure 1: 1 is the blank control, and 2 is the African swine fever virus DNA primase gene DNA. The results show that: no S-shaped amplification curve appears in the blank control, and an S-shaped amplification curve appears in the FAM channel using the African swine fever virus DNA primase gene DNA as a template, indicating that the kit provided by the invention can quickly and accurately detect African swine fever virus DNA. swine fever virus.

Table 2 Fluorescence quantitative PCR reaction system

Third, the specificity of fluorescent quantitative detection methods

Using genomic DNA of porcine pseudorabies virus, porcine circovirus type 2, Erysipelas suis, Actinobacillus pleuropneumoniae, serotype 5 Haemophilus parasuis, serotype 2 Streptococcus suis as the template detection system. sex. The reaction system is shown in Table 2. The reaction conditions are incubation at 37°C for 2 min; pre-denaturation at 95°C for 5 min; 95°C, 10s; 60°C, 30s, 45 cycles. The obtained results are shown in Figure 2: wherein: 1 is the detection result of the serotype 5 Haemophilus parasuis genomic DNA as the template; 2 is the detection result of the serotype 2 Streptococcus suis genomic DNA as the template; 3 is the detection result of the serotype 2 Streptococcus suis genomic DNA. The detection result of Erysipelas suis genomic DNA as the template; 4 is the detection result of Actinobacillus pleuropneumoniae genomic DNA as the template; 5 is the detection result of the porcine pseudorabies virus genomic DNA as the template; 6 is the detection result of porcine circovirus The detection results of type 2 genomic DNA as the template; 7 the detection results of the African swine fever virus DNA primase gene DNA (1.2×10 ³ copies) as the template; 8 is the blank control; serotype 5 Haemophilus parasuis, serum The genomic DNA of type 2 Streptococcus suis, Erysipelas suis, Actinobacillus pleuropneumoniae, porcine pseudorabies virus, and porcine circovirus type 2 were used as templates, respectively, and no S-type amplification curve appeared. The African swine fever virus DNAprimase The gene DNA as the template showed an S-shaped amplification curve in the FAM channel. The results showed that the specificity of the detection system was good, and it could specifically identify and detect African swine fever virus.

Fourth, the sensitivity of fluorescence quantitative PCR detection method

Take 10 μL of African swine fever virus DNA primase gene DNA at a concentration of 0.4 ng/μL (1.2×10 ⁵ copies), add 80 μL of sterilized pure water for 10-fold dilution, take 10 μL of this 10-fold dilution, and add 90 μL in turn 10-fold gradient dilution was carried out in sterilized pure water, and 2 μL of the diluted sample solution was taken for fluorescence quantitative PCR detection (the reaction system is shown in Table 2; except that the template changed, the reaction conditions were all incubated at 37 °C for 2 min; pre-denatured at 95 °C) 5min; 95°C, 10s; 60°C, 30s, 45 cycles). The obtained results are shown in Figure 3: 1 is 1.2×10 ⁵ copies of African swine fever virus DNA primase gene; 2 is 1.2×10 ⁴ copies of African swine fever virus DNA primase gene; 3 is 1.2×10 ³ copies of African swine fever virus DNA primase gene 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 DNA primase 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 African swine fever virus DNA primase gene; 8 is blank control. A S-shaped amplification curve appeared in the FAM channel using the ASFV DNA primase gene DNA at dilutions 1-5 as the template, and no S appeared when the ASFV DNA primase gene DNA at dilutions 6-8 was used as the template The results showed that the method could detect at least 12 copies of African swine fever virus DNAprimase gene DNA.

The foregoing specification illustrates and describes several preferred embodiments of the invention, but as previously mentioned, it should be understood that the invention is not limited to the form disclosed herein and should not be construed as an exclusion of other embodiments, but may be used in a variety of other Combinations, modifications and environments are possible within the scope of the inventive concepts described herein, from the above teachings or from skill or knowledge in the relevant fields. However, modifications and changes made by those skilled in the art do not depart from the spirit and scope of the invention, and should all fall within the protection scope of the appended claims of the invention.

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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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