CN112094950A - Primer group, kit, method and application for detecting African swine fever virus wild strain and gene deletion strain - Google Patents

Abstract

The invention provides a primer group, a kit, a method and application for detecting a wild strain and a gene deletion strain of African swine fever virus, relates to the technical field of virus nucleic acid detection, and provides a primer group for detecting the wild strain and the gene deletion strain of the African swine fever virus for distinguishing the wild strain and the deletion type vaccine strain of the African swine fever virus, wherein the primer group comprises a primer for detecting a VP72 gene, a primer for detecting an MGF360-14L gene and a primer for detecting a CD2V gene. The primer sets provided by the invention are mutually free of interference, the result obtained by amplification has the advantages of accuracy and reliability, high sensitivity, good repeatability, capability of identifying the African swine fever wild strain and the gene-deletion vaccine strain by the same detection system and the like, so that whether a swinery is infected with the African swine fever wild strain or inoculated with the African swine fever gene-deletion vaccine can be accurately and effectively judged, and the loss of farmers is greatly reduced.

Description

Primer group, kit, method and application for detecting African swine fever virus wild strain and gene deletion strain

Technical Field

The invention relates to the technical field of virus nucleic acid detection, in particular to a primer group, a kit, a method and application for detecting a wild strain and a gene deletion strain of African swine fever virus.

Background

African Swine Fever (ASF) is an acute, virulent and highly contagious infectious disease, the morbidity and mortality can reach 100 percent, and the African Swine fever is classified as an animal epidemic disease in China. The first African Swine fever epidemic situation in China is diagnosed in 2018, 8 months and 3 days, and quickly spreads to most regions of the country, thereby causing serious threat to the pig raising industry, and meanwhile, African Swine Fever Virus (ASFV) which causes the epidemic situation is well known in China.

The African swine fever virus is the only member of the African swine fever virus family and is also the only arbovirus DNA virus, the genome has the full length of 170-190kb, contains 151 open reading frames and can code 150-200-protein. The African swine fever virus can be divided into 24 genotypes, and the African swine fever virus discovered in China at this time belongs to genotype II. Published papers on Science by the society of sciences, the society of biophysics, the society of auspicious and the society of agriculture, harbourne, the society of veterinary sciences, in 2019, at 18.10.18.C., revealed unique 5-layer (outer membrane, capsid, double-layer inner membrane, core shell and genome) structural features of african swine fever virus, and virus particles comprising more than 3 ten thousand protein molecules assembled into spherical particles with a diameter of about 260 nm.

After the epidemic situation of the African swine fever occurs, the control of the epidemic situation mainly depends on killing, but the control is not the most ideal epidemic situation prevention and control means, so that the development of the vaccine capable of effectively and safely preventing the epidemic situation is very important. Under the efforts of Chinese researchers, the development of the domestic African swine fever vaccine is very rapid. Based on the previous research, the Chinesemedicine institute of Harbin veterinary institute introduced in CN110093324A, separated Chinese epidemic strains (Pig/CN/HLJ/2018) are subjected to MGF360-505R single gene deletion or MGF360-505R and CD2V double gene deletion to obtain two gene deletion type vaccine strains (China typical culture Collection accession number: CCTCC NO: V201925 and CCTCC NO: V201924), 6 genes including MGF360-505R sequence and MGF505-3R in the two gene deletion type strains are subjected to a virus test on pigs immunized by the two gene deletion type swine fever virus relative to a full-length sequence at position 27942 35500, the sequences comprise MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R and MGF505-3R, the immunized pigs do not show the symptom of African disease, the survival rate can reach 100 percent, which shows that the two gene deletion strains have very important significance as vaccines, the toxicity of the strains is obviously weakened after the gene deletion, the attack of the African swine fever wild strains can be successfully immunized, and the safety and the reliability are realized. In addition, the SY18 isolate of the African swine fever is prepared into a gene-deficient attenuated vaccine strain by Chenhong et al in CN 110551695A of Shanghai veterinary medical institute of Chinese agricultural academy of sciences, the deficient genes are four types, namely CD2V, MGF360-12L, MGF360-13L and MGF360-14L, the attenuated strain is used for inoculating piglets for 28 days, an African swine fever virus parent is used for carrying out a virus challenge test to observe immune pig groups, and no abnormal clinical symptoms are seen, so that the four-gene-deficient attenuated strain can be used as a vaccine candidate for preventing the African swine fever. Researchers at the harbin veterinary institute of chinese academy of agricultural sciences are in "china science: in an article 'A seven-gene-deleted African swine fever virus is safe and effective as a live inactivated vaccine in pigs' published in Life sciences, a series of recombinant viruses with different gene deletions are constructed by using a homologous recombination technology based on a first African swine fever virus isolate strain Pig/HLJ/2018 in China, a virus (HLJ/18-7GD) with 7 genes (including MGF505-1R, MGF505-2R, MGF505-3R, MGF-12L, MGF-13L, MGF-14L and CD2V genes) deletions is screened out by carrying out pathogenicity, immunogenicity and immunoprotection tests in pigs, the virus is injected into pigs without specific pathogens for 21 days and then is subjected to challenge virus survival rate tests, the immunity of the pigs reaches 100 percent, and therefore, the vaccine serving as a swine fever virus gene deletion inactivated vaccine meets the safety standard of African swine fever virus inactivation vaccine, can provide effective immune protection for the lethal attack of the African swine fever virulent virus, has industrial application prospect, and provides an important technical means for the effective prevention and control of the African swine fever epidemic situation in China and related countries.

At present, enzyme-linked immunosorbent assay, common PCR detection method, real-time fluorescence quantitative PCR detection method and the like are used for detecting African swine fever virus, wherein the real-time fluorescence quantitative PCR detection method is mainly realized by designing primer pairs and Taqman probes on VP72, and the operation is simple and reliable. However, if the candidate vaccine strain is used as an African swine fever vaccine and then a swine herd is detected, it is difficult to determine whether the swine herd is inoculated with the African swine fever gene deletion type vaccine strain or infected with the wild African swine fever strain. Therefore, the detection method capable of distinguishing the African swine fever virus wild strain and the deletion type vaccine strain is found, the loss of farmers can be reduced, and the detection method has important significance.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first objective of the invention is to provide a primer group for detecting a wild strain and a gene deletion strain of African swine fever virus, so as to at least alleviate one of the technical problems in the prior art.

The second purpose of the invention is to provide a kit for detecting the African swine fever virus wild strain and the gene deletion strain.

The third purpose of the invention is to provide a method for detecting the African swine fever virus wild strain and the gene deletion strain.

The fourth purpose of the invention is to provide the application of the primer group, the kit or the method in detecting the African swine fever virus wild strain and the gene deletion strain.

The invention provides a primer group for detecting a wild strain and a gene deletion strain of African swine fever virus, which comprises a primer for detecting a VP72 gene, a primer for detecting an MGF360-14L gene and a primer for detecting a CD2V gene:

the primer for detecting the VP72 gene has the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or the nucleotide sequence with at least 85% of identity with SEQ ID NO.1 and/or SEQ ID NO. 2;

the primer for detecting the MGF360-14L gene has nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5, or nucleotide sequences with at least 85% of identity with SEQ ID NO.4 and/or SEQ ID NO. 5;

the primer for detecting the CD2V gene has the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8, or the nucleotide sequence which has at least 85% of identity with the SEQ ID NO.7 and/or the SEQ ID NO. 8.

Further, the primer group also comprises a probe for detecting a VP72 gene, a probe for detecting an MGF360-14L gene and a probe for detecting a CD2V gene;

the probe for detecting the VP72 gene has a nucleotide sequence shown as SEQ ID NO.3, or a nucleotide sequence with at least 85% of identity with SEQ ID NO. 3;

the probe for detecting the MGF360-14L gene has a nucleotide sequence shown as SEQ ID NO.6 or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO. 6;

the probe for detecting the CD2V gene has a nucleotide sequence shown as SEQ ID NO.9 or a nucleotide sequence with at least 85% of identity with the SEQ ID NO. 9.

Furthermore, the probe for detecting the VP72 gene, the probe for detecting the MGF360-14L gene and the probe for detecting the CD2V gene respectively contain a fluorescence quenching group and a fluorescence reporter group which are different from each other at two ends independently;

preferably, the fluorescent reporter group comprises FAM, HEX or ROX; the probe for detecting the VP72 gene preferably contains FAM, the probe for detecting the MGF360-14L gene preferably contains HEX, and the probe for detecting the CD2V gene preferably contains ROX;

preferably, the fluorescence quenching group comprises BHQ1 or BHQ 2.

The invention also provides a kit for detecting the African swine fever virus wild strain and the gene deletion strain, and the kit comprises the primer group.

Further, the kit also comprises a positive control substance and/or a negative control substance.

Further, the positive control includes a plasmid containing a part or all of the VP72 gene, a plasmid containing a part or all of the MGF360-14L gene, and a plasmid containing a part or all of the CD2V gene;

preferably, the negative control comprises a solution for diluting the positive control, preferably a TE solution.

The invention also provides a method for detecting the African swine fever virus wild strain and the gene deletion strain, which comprises the steps of taking the genome DNA of a sample to be detected as a template, carrying out PCR reaction by using the primer group or the kit, and judging whether the sample to be detected contains the African swine fever virus wild strain or the gene deletion strain according to a specific fragment or an amplification curve contained in an amplification product:

if the amplification product contains specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene, the sample to be detected contains a wild strain of African swine fever virus; if the amplification product does not contain specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene at the same time, the sample to be detected does not contain the wild strain and the gene deletion strain of the African swine fever virus; if the amplification product contains a specific fragment or an amplification curve of the VP72 gene and does not contain a specific fragment or an amplification curve of the MGF360-14L gene and the CD2V gene, the sample to be detected contains MGF360-505R and CD2V double-gene deletion strains; if the amplification product contains specific fragments or amplification curves of the VP72 gene and the MGF360-14L gene and does not contain specific fragments or amplification curves of the CD2V gene, the sample to be detected contains a CD2V gene deletion strain; if the amplification product contains specific fragments or amplification curves of the VP72 gene and the CD2V gene, and does not contain specific fragments or amplification curves of the MGF360-14L gene, the MGF360-505R single gene deletion strain is contained in the sample to be detected.

Further, the PCR is triple fluorescence quantitative PCR, and the reaction conditions of the triple fluorescence quantitative PCR are as follows: pre-denaturation at 92-98 deg.C for 2-4 min; denaturation at 92-98 deg.C for 8-12s, and annealing at 58-62 deg.C for 12-18s for 42-45 cycles;

preferably, the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 15s for 45 cycles.

Further, the probe for detecting the VP72 gene contains FAM, the probe for detecting the MGF360-14L gene contains HEX, and the probe for detecting the CD2V gene contains ROX;

when the FAM, the HEX and the ROX are not amplified, the sample to be detected does not contain the African swine fever virus wild strain and the gene deletion strain;

when the FAM, the HEX and the ROX have amplification curves and the Ct value is less than 39, the sample to be detected contains the African swine fever virus wild strain;

when FAM has an amplification curve and Ct value is less than 39, but neither HEX nor ROX is amplified, MGF360-505R and CD2V double-gene deletion strains exist in the sample to be detected;

when FAM and HEX have amplification curves and Ct value is less than 39 but ROX has no amplification, CD2V gene deletion strain exists in the sample to be detected;

when FAM and ROX have amplification curves and Ct value is less than 39, but HEX has no amplification, MGF360-505R single gene deletion strain exists in the sample to be detected.

In addition, the invention also provides application of the primer group, the kit or the method in detecting the African swine fever virus wild strain and the gene deletion strain.

The invention provides a primer group for detecting the African swine fever virus wild strain and a gene deletion strain for distinguishing the African swine fever virus wild strain and the deletion type vaccine strain, which comprises a primer for detecting a VP72 gene, a primer for detecting an MGF360-14L gene and a primer for detecting a CD2V gene. By applying the primer group provided by the invention, the wild strain of African swine fever, the MGF360-505R single-gene deletion strain and the MGF360-505R and CD2V double-gene deletion strain can be distinguished in the same reaction tube, so that reaction reagents and reaction time are saved, and the detection efficiency is obviously improved. Meanwhile, the primer sets provided by the invention are mutually free of interference, the amplification result has the advantages of accuracy and reliability, high sensitivity, good repeatability, capability of identifying the African swine fever wild strain and the gene deletion type vaccine strain by the same detection system and the like, so that whether a swinery is infected with the African swine fever wild strain or inoculated with the African swine fever gene deletion type vaccine can be accurately and effectively judged, and the loss of farmers is greatly reduced.

The invention provides a method for detecting a wild strain and a gene deletion strain of African swine fever virus by optimizing reaction conditions. According to the method provided by the invention, the primer group provided by the invention is used for carrying out PCR detection on a sample to be detected, accurate molecular identification of the African swine fever virus wild strain and the deletion type vaccine strain is realized according to different detection results, whether the sample to be detected is an immunized African swine fever wild strain or an infected African swine fever wild strain is analyzed, and the method has the advantages of simplicity and convenience in operation, rapidness, high sensitivity, high specificity, high throughput and the like. Meanwhile, no method for identifying the wild strain and the vaccine strain of the African swine fever is established in China at present, so that the invention provides a technical thought for identifying the African swine fever virus in China and strongly supports the development of the pig raising industry in China.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram of the triple fluorescence PCR optimization results of African swine fever virus wild strains and gene deletion strains provided by the embodiment of the invention;

FIG. 2 is a diagram showing the result of triple fluorescence PCR specificity verification of the African swine fever virus wild strain and the gene deletion strain provided by the embodiment of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

Based on the important significance of the gene deletion strain in preparing the vaccine, the invention provides a primer group for detecting the African swine fever virus wild strain and the gene deletion strain on one hand, and is characterized by comprising a primer for detecting the VP72 gene, a primer for detecting the MGF360-14L gene and a primer for detecting the CD2V gene:

the primer for detecting the VP72 gene has the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or the nucleotide sequence with at least 85% of identity with SEQ ID NO.1 and/or SEQ ID NO. 2;

the primer for detecting the MGF360-14L gene has nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5, or nucleotide sequences with at least 85% of identity with SEQ ID NO.4 and/or SEQ ID NO. 5;

the primer for detecting the CD2V gene has the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8, or the nucleotide sequence which has at least 85% of identity with the SEQ ID NO.7 and/or the SEQ ID NO. 8.

By applying the primer group provided by the invention, a plurality of different target genes can be detected in the same reaction tube, reaction reagents and reaction time are saved, and the detection efficiency is obviously improved. Meanwhile, the primer group provided by the invention has strong specificity, high sensitivity and stable amplification capacity, can simply, quickly and accurately identify and detect the African swine fever virus wild strain and the gene deletion strain in a sample to be detected, further accurately and effectively judges whether a swinery is infected with the African swine fever wild strain or inoculated with the African swine fever gene deletion type vaccine, and greatly reduces the loss of farmers.

Wherein "identity" refers to similarity between nucleotide sequences, including nucleotide sequences having at least 85% (e.g., but not limited to, 85%, 88%, 90%, 92%, 95%, 98%, 99% or more) identity to the nucleotide sequences set forth in SEQ ID No.1, 2, 4, 5, 7, 8 of the present invention. When the nucleotide sequences shown in SEQ ID NO.1, 2, 4, 5, 7 and 8 are selected as the primers in the primer group provided by the invention, the primer group has stronger specificity and higher sensitivity.

"nucleotide sequence having at least 85% identity with SEQ ID No.1 and/or SEQ ID No. 2" means: for example, the primers used for detecting the VP72 gene can be SEQ ID NO.1 and SEQ ID NO.2, or a nucleotide sequence having at least 85% identity with SEQ ID NO.2 and SEQ ID NO.1, or a nucleotide sequence having at least 85% identity with SEQ ID NO.1 and a nucleotide sequence having at least 85% identity with SEQ ID NO. 2. The primers for detecting MGF360-14L gene and the primers for detecting CD2V gene are the same.

In addition, it should be noted that there are many gene deletion modes for African swine fever gene deletion vaccine strains, such as MGF360-505R single gene deletion or MGF360-505R and CD2V double gene deletion vaccine strains developed by the high school of veterinary medicine of Harbin institute of agriculture, academy of sciences, China which are more commonly used at present, wherein the MGF360-505R sequence is at position 27942-35500 relative to the full-length sequence, and the sequence includes 6 genes including MGF505-1R, MGF-12L, MGF-360-3613 360-L, MGF-505-2R and MGF 505-3R. Therefore, when MGF360-14L is deleted, the MGF360-505R gene comprising it is also deleted. The primer group provided by the invention can detect single gene deletion of MGF360-505R, single gene deletion of CD2V or double gene deletion of MGF360-505R and CD2V by detecting VP72 gene, MGF360-14L and CD2V gene, and can also distinguish seven gene deletion strains containing MGF360-14L and CD 2V. For example, the separated strain of the African swine fever SY18 is prepared into a gene-deleted attenuated vaccine strain by the Chenhong army of the institute of veterinary medicine at the Shanghai of Chinese academy of agricultural sciences, wherein the deleted genes include four types, namely CD2V, MGF360-12L, MGF360-13L and MGF 360-14L. The primer group provided by the invention can detect MGF360-505R single gene deletion, CD2V single gene deletion or MGF360-505R and CD2V double gene deletion by detecting VP72 gene, MGF360-14L and CD2V gene, and can simultaneously distinguish four-gene deletion strain containing MGF360-14L and CD2V as vaccine candidate strain.

The primer set provided by the invention is suitable for various PCR reactions. For example, when conventional PCR is used, whether the test sample contains the VP72 gene, MGF360-14L gene or CD2V gene can be determined based on the presence or absence of an amplification product band and the size of the fragment of the amplification product. When the fluorescence quantitative PCR is used, whether the VP72 gene, the MGF360-14L gene or the CD2V gene is contained in the sample to be detected can be judged according to the fluorescence signal in the amplification product and the amplification curve. The specific characterization method of the fluorescent quantitative PCR is not limited, and typical characterization methods are dye methods or probe methods commonly used in the art. In order to further improve the specificity of the fluorescent quantitative PCR reaction, a probe method is preferably used. Based on this, in some preferred embodiments, the primer set further comprises a probe for detecting the VP72 gene, a probe for detecting the MGF360-14L gene, and a probe for detecting the CD2V gene.

In some preferred embodiments, the probe for detecting the VP72 gene has a nucleotide sequence as shown in SEQ ID No.3, or a nucleotide sequence at least 85% identical to SEQ ID No. 3;

the probe for detecting the MGF360-14L gene has a nucleotide sequence shown as SEQ ID NO.6 or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO. 6;

the probe for detecting the CD2V gene has a nucleotide sequence shown as SEQ ID NO.9 or a nucleotide sequence with at least 85% of identity with the SEQ ID NO. 9.

The probe improved by the embodiment is designed aiming at all or part of sequences of the VP72 gene, the MGF360-14L gene and the CD2V gene, has no cross reaction with other genes, and also has the advantages of strong specificity and high sensitivity. "identity" includes nucleotide sequences having at least 85% (e.g., can be, but is not limited to, 85%, 88%, 90%, 92%, 95%, 98%, 99% or more) identity to the nucleotide sequences set forth in SEQ ID nos. 3, 6, and 9 described in the present embodiment. When the nucleotide sequences shown in SEQ ID Nos. 3, 6 and 9 are selected as the probe provided in the present embodiment, they have stronger specificity and higher sensitivity.

In some preferred embodiments, the probe for detecting the VP72 gene, the probe for detecting the MGF360-14L gene, and the probe for detecting the CD2V gene each independently comprise a fluorescence quenching group and a fluorescence reporter group different from each other at both ends. The specific choice of the fluorescence reporter group and the fluorescence quencher group contained in the probe is not limited in this embodiment, and all groups conventionally available in the art can be used as the linking group of the probe in this embodiment.

Preferably, the fluorescent reporter group comprises FAM, HEX or ROX; the probe for detecting the VP72 gene preferably contains FAM, the probe for detecting the MGF360-14L gene preferably contains HEX, and the probe for detecting the CD2V gene preferably contains ROX;

preferably, the fluorescence quenching group comprises BHQ1 or BHQ 2.

Based on the same inventive concept of the primer set provided by the invention, the invention also provides a kit for detecting the African swine fever virus wild strain and the gene deletion strain, so that the kit for detecting the African swine fever virus wild strain and the gene deletion strain provided by the invention has the overall beneficial effects with the primer set provided by the invention, and the details are not repeated herein.

In some preferred embodiments, the kit further comprises a positive control and/or a negative control. "and/or" means that the kit provided by the invention can contain a positive control substance alone, or a negative control substance alone, or both the positive control substance and the negative control substance. By arranging the positive control substance and the negative control substance, the negative or false positive result in the detection can be effectively avoided, and the detection accuracy is further ensured.

Preferably, the positive control includes a plasmid containing part or all of VP72 gene, a plasmid containing part or all of MGF360-14L gene, and a plasmid containing part or all of CD2V gene; preferably, a mixture of plasmids (AS-VP72, AS-CD2V and AS-MGF360-14L) containing partial sequences of VP72 and MGF360-14L, CD2V genes is used.

Preferably, the negative control includes a solution for diluting the positive control to achieve a controlled variable to ensure consistency of the test results. TE solution is preferably used as a positive control solution and a negative control.

According to the third aspect of the present invention, there is also provided a method for detecting a wild strain and a gene-deleted strain of african swine fever virus: the genome DNA of a sample to be detected is used as a template, the primer group or the kit provided by the invention is used for PCR reaction, and whether the sample to be detected contains the African swine fever virus wild strain or the gene deletion strain is judged according to a specific segment or an amplification curve contained in an amplification product:

if the amplification product contains specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene, the sample to be detected contains a wild strain of African swine fever virus; if the amplification product does not contain specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene at the same time, the sample to be detected does not contain the wild strain and the gene deletion strain of the African swine fever virus; if the amplification product contains a specific fragment or an amplification curve of a VP72 gene and does not contain a specific fragment or an amplification curve of an MGF360-14L gene and a CD2V gene, a sample to be detected contains MGF360-505R and CD2V double-gene deletion strains, (when detecting MGF360-505R single-gene deletion or MGF360-505R and CD2V double-gene deletion vaccine strains developed by a high team of Harbin veterinary institute of Chinese academy of agriculture sciences, seven gene deletion strains including MGF360-14L and CD2V in the sample to be detected can be further explained, when detecting that a separated strain of African swine fever SY18 in Shanghai veterinary institute of China academy of agriculture sciences is prepared into a gene deletion attenuated vaccine strain, four gene deletion strains including MGF360-14L and CD2V in the sample to be detected can be also explained); if the amplification product contains specific fragments or amplification curves of the VP72 gene and the MGF360-14L gene and does not contain specific fragments or amplification curves of the CD2V gene, the sample to be detected contains a CD2V gene deletion strain; if the amplification product contains specific fragments or amplification curves of the VP72 gene and the CD2V gene, and does not contain specific fragments or amplification curves of the MGF360-14L gene, the MGF360-505R single gene deletion strain is contained in the sample to be detected.

According to different detection results, accurate molecular identification of the African swine fever virus wild strain and the deletion type vaccine strain is realized, whether a sample to be detected is an immunized African swine fever wild strain or an infected African swine fever wild strain is analyzed, and the method has the advantages of simplicity and convenience in operation, rapidness, high sensitivity, high specificity, high throughput and the like. Meanwhile, no method for identifying the wild strain and the vaccine strain of the African swine fever is established in China at present, so that the invention provides a technical thought for identifying the African swine fever virus in China and strongly supports the development of the pig raising industry in China.

It should be noted that, since the primer set provided by the present invention is applicable to various types of PCR reactions, there are correspondingly different methods for determining results corresponding to different types of PCR reactions. For example, when conventional PCR is used, whether the test sample contains the VP72 gene, MGF360-14L gene or CD2V gene can be determined based on the presence or absence of an amplification product band and the size of the fragment of the amplification product. When the fluorescence quantitative PCR is used, whether the VP72 gene, the MGF360-14L gene or the CD2V gene is contained in the sample to be detected can be judged according to the fluorescence signal in the amplification product and the amplification curve. Different PCR reaction types and corresponding different result judgment methods can be matched with the primer group or the kit provided by the invention to realize accurate molecular identification of the African swine fever virus wild strain and the deletion type vaccine strain.

In some preferred embodiments, the PCR is triple fluorescence quantitative PCR, and the reaction conditions of the triple fluorescence quantitative PCR include at least one of the following conditions: pre-denaturation at 92-98 deg.C for 2-4 min; denaturation at 92-98 deg.C for 8-12s, and annealing at 58-62 deg.C for 12-18s for 42-45 cycles;

the preferable reaction condition is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 15s for 45 cycles.

The PCR reaction conditions are important factors influencing the PCR specificity, and the reaction conditions of the triple fluorescence quantitative PCR are further optimized, so that the specificity is stronger and the result accuracy is higher when the method for detecting the African swine fever virus wild strain and the gene deletion strain provided by the invention is used for identifying and detecting the African swine fever virus wild strain and the deletion type vaccine strain.

In some preferred embodiments, the probe for detecting the VP72 gene comprises FAM, the probe for detecting the MGF360-14L gene comprises HEX, and the probe for detecting the CD2V gene comprises ROX;

when the FAM, the HEX and the ROX are not amplified, the sample to be detected does not contain the African swine fever virus wild strain and the gene deletion strain;

when the FAM, the HEX and the ROX have amplification curves and the Ct value is less than 39, the sample to be detected contains the African swine fever virus wild strain;

when FAM has an amplification curve and Ct value is less than 39 but both HEX and ROX are not amplified, MGF360-505R and CD2V double-gene deletion strains exist in a sample to be detected (when MGF360-505R single-gene deletion or MGF360-505R and CD2V double-gene deletion vaccine strains researched and developed by Harbin veterinary institute of Chinese academy of agricultural sciences are detected, the gene deletion strains containing MGF360-14L and CD2V in the sample to be detected can be further explained, and when seven African swine fever SY18 isolates are prepared into gene deletion attenuated vaccine strains by Chenhong Jun et al of Shanghai veterinary institute of Chinese academy of agricultural sciences, the four-gene deletion strains containing MGF360-14L and CD2V in the sample to be detected can be also explained);

when FAM and HEX have amplification curves and Ct value is less than 39 but ROX has no amplification, CD2V gene deletion strain exists in the sample to be detected;

when FAM and ROX have amplification curves and Ct value is less than 39, but HEX has no amplification, MGF360-505R single gene deletion strain exists in the sample to be detected.

In addition, when the positive control substances (AS-VP72, AS-CD2V and AS-MGF360-14L) provided by the invention are detected by using a triple fluorescence quantitative PCR method, Ct values of FAM, HEX and ROX channels are all 25 +/-2.

When the negative control provided by the invention is detected by using a triple fluorescence quantitative PCR method, all channels are not amplified.

In addition, the invention also provides application of the primer group, the kit or the method in detecting the African swine fever virus wild strain and the gene deletion strain. The primer group, the kit or the method provided by the invention are used for identifying and detecting the African swine fever virus wild strain and the gene deletion strain, and have the advantages of short detection time and low cost.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagent information used in the examples of the present invention is as follows:

example 1

1. Primer Probe design

The inventor downloads a large number of gene sequences containing VP72 or MGF360-14L and CD2V of African swine fever virus in GenBank, finds out corresponding primer probes designed by the highly conserved regions of the three genes through sequence comparison, and the sequences are as follows:

SEQ ID NO：1：5’-TCGCCGAAGGGAATGGATA-3’

SEQ ID NO：2：5’-CCMATKACGGACGCAACGT-3’

SEQ ID NO：3：5’-FAM-GAGGGAATAGCAAGGTTCACGTTCTCA-BHQ1-3’

SEQ ID NO：4：5’-CGGTTTCCAARAGTGGATTATGAC-3’

SEQ ID NO：5：5’-AAATCCTGAATATGGRCTTATACGT-3’

SEQ ID NO：6：5’-HEX-ACATGTCATAACCCAGCAGAACTCCTGC-BHQ1-3’

SEQ ID NO：7：5’-ACCACCTGAATCTAATGAAGAAGAACA-3’

SEQ ID NO：8：5’-GGTGGAGGACACGGTTTAGGTAAG-3’

SEQ ID NO：9：5’-ROX-CATGATGACACCACTTCCATACATGAACCA-BHQ2-3’

wherein, SEQ ID NO: 1 to SEQ ID NO: 3 is specific primer and probe for detecting VP72, SEQ ID NO: the 5 'and 3' ends of 3 are labeled as FAM and BHQ1, respectively;

SEQ ID NO: 4 to SEQ ID NO: 6 is a specific primer and probe for detecting MGF360-14L, SEQ ID NO: the 5 'and 3' ends of 6 are labeled HEX and BHQ1, respectively;

SEQ ID NO: 7 to SEQ ID NO: 9 is specific primer and probe for detecting CD2V, SEQ ID NO: the 5 'and 3' ends of 9 are labeled ROX and BHQ2, respectively.

2. Preparation of Positive control

Partial sequences of genes of African swine fever virus VP72 and MGF360-14L, CD2V are respectively connected with PUC57, escherichia coli DH5 alpha is transformed, a resistant plate is coated, then a single clone is screened, and the positive strains containing the three gene fragments are respectively obtained after the sequencing is successful. After the corresponding strains were expanded and cultured, the positive plasmids were extracted using the procedures described in Biospin plasmid DNA miniprep kit of Hangzhou Bori science and technology, Inc., and the extracted positive plasmids were named AS-VP72, AS-CD2V and AS-MGF360-14L, respectively. Mixing AS-VP72, AS-CD2V and AS-MGF360-14L according to a certain proportion, and using SEQ ID NO: 1 to SEQ ID NO: 9, performing fluorescence PCR amplification, and when the Ct value of each channel is 25 +/-2, the positive plasmid mixed solution is a positive control, and each tube is separately filled with 500 mu L of the positive control and stored at-20 ℃ for later use.

3. Preparation of negative control

The negative control is TE solution for diluting the positive control, and each tube is divided into 500 μ L for storage at-20 deg.C.

Example 2 Assembly of Triplex fluorescent PCR detection kit for African swine fever virus wild strains and gene-deleted strains

1. PCR reaction solution

The kit comprises three pairs of primers and three corresponding TaqMan probes, wherein the sequences of the upstream primer and the downstream primer for detecting the VP72 gene and the probe are respectively SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the sequences of an upstream primer and a downstream primer for detecting MGF360-14L genes and a probe are respectively SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification; the sequences of an upstream primer and a downstream primer for detecting the CD2V gene and a probe are respectively SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: shown at 9.

2. Positive control

A positive plasmid mixture containing AS-VP72, AS-CD2V, and AS-MGF360-14L, using the nucleic acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 9, performing fluorescence PCR amplification, wherein the Ct value of each channel is 25 +/-2.

3. And (5) a negative control product.

TE solution for diluting positive control, and a primer probe set in the kit is used for carrying out fluorescence PCR detection, wherein each channel has no amplification.

4. Description of the invention

The kit contains a specification which comprises kit components, a use method, result judgment and the like.

5. External packing

The external package is packaged by using a specific packaging material and a specific packaging mode of PCR products of Hangzhou Bori science and technology limited company.

Example 3 African swine fever virus wild strain and gene deletion strain triple fluorescence PCR detection method

1. Design verification of primer Probe in example 1

2. Extraction of nucleic acids from a sample: simplyp virus DNA/RNA extraction kit from Hangzhou Bori science and technology, Inc. can be used, and the extraction steps refer to the kit instructions.

3. Triple fluorescent PCR amplification was performed as follows

3.1 extracting the virus nucleic acid in the sample according to the instruction of the virus nucleic acid extraction kit;

3.2 PCR reaction solution containing three pairs of primer probe sets for specifically detecting VP72, CD2V, MGF360-14L genes was dispensed in 20. mu.L per reaction tube, and 5. mu.L of the sample nucleic acid extracted in 3.1 was added for fluorescence PCR detection, which was repeated once per sample.

3.3 the amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 15s, for 45 cycles; fluorescence signals of FAM, HEX and ROX channels were collected at 60 ℃.

3.4 conditions effective for the experiments: in the same experiment, Ct values of three channels of FAM, HEX and ROX which simultaneously meet the requirements of positive control substances are all smaller than 30, and three channels of negative control substances are not amplified, so that the experiment is effective, otherwise, the experiment is invalid.

3.5 principle of result judgment: when FAM, HEX and ROX are not amplified, the result shows that the sample has no African swine fever virus wild strain or African swine fever virus gene deletion strain, and the sample is a negative sample;

when the amplification results of FAM, HEX and ROX all have typical amplification curves and the Ct value is less than 39, the African swine fever wild strain exists in the sample;

when the amplification result of FAM has a typical amplification curve and Ct value is less than 39 but both HEX and ROX are not amplified, the result shows that MGF360-505R and CD2V double-gene deletion strains exist in the sample (when MGF360-505R single-gene deletion or MGF360-505R and CD2V double-gene deletion vaccine strains developed by the Shimeji veterinary institute of Harbin institute of China are detected, seven gene deletion strains including MGF360-14L and CD2V in the sample to be detected can be further indicated, and when the SEY 18 isolate of Naja veterinary institute of China Shanghai veterinary institute of China is prepared into a gene deletion attenuated vaccine strain, four gene deletion strains including MGF360-14L and CD2V in the sample to be detected can be further indicated);

when the amplification results of FAM and HEX have typical amplification curves and the Ct value is less than 39 but ROX is not amplified, indicating that a strain containing CD2V gene deletion exists in the sample;

when the amplification results of FAM and ROX have typical amplification curves and the Ct value is less than 39, but HEX is not amplified, the MGF360-505R single gene deletion strain exists in the sample.

The triple fluorescence PCR optimization results of the African swine fever virus wild strain and the gene deletion strain are shown in figure 1, wherein A: VP72 (FAM); b: MGF360-14L (HEX); c: CD2V (ROX); d: and (5) negative control.

Example 4 specificity verification experiment

Detecting and analyzing the result according to the triple fluorescence PCR detection method described in the embodiment 3 for African swine fever wild strain, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine infectious pleuropneumonia virus, porcine pseudorabies virus, porcine epidemic diarrhea virus, toxoplasma, porcine rotavirus, positive plasmid AS-VP72, positive plasmid AS-MGF360-14L, positive plasmid AS-CD2V, positive control and negative control, wherein the negative and positive controls meet the requirements, therefore, the test is effective, the wild strains FAM, HEX and ROX of African swine fever have Ct values, the positive plasmid AS-VP72 only has the Ct value in the FAM channel, the positive plasmid AS-MGF360-14L only has the Ct value in the HEX, the positive plasmid AS-CD2V only has the Ct value in the ROX, and no amplification exists in samples containing other viruses, so that the detection kit and the detection method have good specificity (the result is shown in figure 2).

Example 5 identification of wild Strain samples and Gene-deleted strains containing African Swine fever Virus

20 blind samples (including a sample containing a wild strain of African swine fever virus and a gene-deficient strain) were identified according to the triple fluorescence PCR assay described in example 3, and the results are shown in Table 1:

TABLE 1 summary of sample test results

Wherein "- -" indicates that the detection result has no Ct value.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

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Claims (10)

1. A primer group for detecting wild strains and gene deletion strains of African swine fever virus is characterized by comprising a primer for detecting VP72 gene, a primer for detecting MGF360-14L gene and a primer for detecting CD2V gene:

the primer for detecting the VP72 gene has the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, or the nucleotide sequence with at least 85% of identity with SEQ ID NO.1 and/or SEQ ID NO. 2;

the primer for detecting the MGF360-14L gene has nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5, or nucleotide sequences with at least 85% of identity with SEQ ID NO.4 and/or SEQ ID NO. 5;

the primer for detecting the CD2V gene has the nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8, or the nucleotide sequence which has at least 85% of identity with the SEQ ID NO.7 and/or the SEQ ID NO. 8.

2. The primer set according to claim 1, further comprising a probe for detecting VP72 gene, a probe for detecting MGF360-14L gene, and a probe for detecting CD2V gene;

the probe for detecting the VP72 gene has a nucleotide sequence shown as SEQ ID NO.3, or a nucleotide sequence with at least 85% of identity with SEQ ID NO. 3;

the probe for detecting the MGF360-14L gene has a nucleotide sequence shown as SEQ ID NO.6 or a nucleotide sequence which has at least 85% of identity with the SEQ ID NO. 6;

the probe for detecting the CD2V gene has a nucleotide sequence shown as SEQ ID NO.9 or a nucleotide sequence with at least 85% of identity with the SEQ ID NO. 9.

3. The primer set according to claim 2, wherein the probe for detecting the VP72 gene, the probe for detecting the MGF360-14L gene, and the probe for detecting the CD2V gene each independently have a fluorescence quencher and a fluorescence reporter different from each other at both ends;

preferably, the fluorescent reporter group comprises FAM, HEX or ROX; the probe for detecting the VP72 gene preferably contains FAM, the probe for detecting the MGF360-14L gene preferably contains HEX, and the probe for detecting the CD2V gene preferably contains ROX;

preferably, the fluorescence quenching group comprises BHQ1 or BHQ 2.

4. A kit for detecting a wild strain and a gene deletion strain of African swine fever virus, which comprises the primer set of any one of claims 1-3.

5. The kit of claim 4, further comprising a positive control and/or a negative control.

6. The kit of claim 5, wherein the positive control comprises a plasmid containing part or all of VP72 gene, a plasmid containing part or all of MGF360-14L gene, and a plasmid containing part or all of CD2V gene;

preferably, the negative control comprises a solution for diluting the positive control, preferably a TE solution.

7. A method for detecting a wild strain or a gene-deleted strain of African swine fever virus, which is characterized in that, a genome DNA of a sample to be detected is used as a template, a PCR reaction is carried out by using the primer group of any one of claims 1 to 3 or the kit of any one of claims 4 to 6, and whether the sample to be detected contains the wild strain or the gene-deleted strain of the African swine fever virus is judged according to a specific fragment or an amplification curve contained in an amplification product:

if the amplification product contains specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene, the sample to be detected contains a wild strain of African swine fever virus; if the amplification product does not contain specific fragments or amplification curves of the VP72 gene, the MGF360-14L gene and the CD2V gene at the same time, the sample to be detected does not contain the wild strain and the gene deletion strain of the African swine fever virus; if the amplification product contains a specific fragment or an amplification curve of the VP72 gene and does not contain a specific fragment or an amplification curve of the MGF360-14L gene and the CD2V gene, the sample to be detected contains MGF360-505R and CD2V double-gene deletion strains; if the amplification product contains specific fragments or amplification curves of the VP72 gene and the MGF360-14L gene and does not contain specific fragments or amplification curves of the CD2V gene, the sample to be detected contains a CD2V gene deletion strain; if the amplification product contains specific fragments or amplification curves of the VP72 gene and the CD2V gene, and does not contain specific fragments or amplification curves of the MGF360-14L gene, the MGF360-505R single gene deletion strain is contained in the sample to be detected.

8. The method of claim 7, wherein the PCR is triple fluorescence quantitative PCR, and the reaction conditions of the triple fluorescence quantitative PCR comprise at least one of the following conditions: pre-denaturation at 92-98 deg.C for 2-4 min; denaturation at 92-98 deg.C for 8-12s, and annealing at 58-62 deg.C for 12-18s for 42-45 cycles;

the preferable reaction condition is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 15s for 45 cycles.

9. The method as claimed in claim 8, wherein the probe for detecting the gene VP72 comprises FAM, the probe for detecting the gene MGF360-505R comprises HEX, and the probe for detecting the gene CD2V comprises ROX;

when the FAM, the HEX and the ROX are not amplified, the sample to be detected does not contain the African swine fever virus wild strain and the gene deletion strain;

when the FAM, the HEX and the ROX have amplification curves and the Ct value is less than 39, the sample to be detected contains the African swine fever virus wild strain;

when FAM has an amplification curve and Ct value is less than 39, but neither HEX nor ROX is amplified, MGF360-505R and CD2V double-gene deletion strains exist in the sample to be detected;

when FAM and HEX have amplification curves and Ct value is less than 39 but ROX has no amplification, CD2V gene deletion strain exists in the sample to be detected;

when FAM and ROX have amplification curves and Ct value is less than 39, but HEX has no amplification, MGF360-505R single gene deletion strain exists in the sample to be detected.

10. Use of the primer set according to any one of claims 1 to 3, the kit according to any one of claims 4 to 6 or the method according to any one of claims 7 to 9 for detecting wild strains and gene-deleted strains of African swine fever virus.

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