CN110872637A

CN110872637A - Reagent for identifying African swine fever gene deletion vaccine, detection method and application

Info

Publication number: CN110872637A
Application number: CN201911227422.1A
Authority: CN
Inventors: 林彦星; 花群俊; 史卫军; 曹琛福; 黄超华; 花群义; 杨俊兴; 张彩虹; 阮周曦
Original assignee: Wuding County Animal Epidemic Prevention And Control Center; Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Current assignee: Wuding County Animal Epidemic Prevention And Control Center; Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2020-03-10

Abstract

The application discloses a reagent for identifying African swine fever gene deletion vaccine, a detection method and application. The reagent of the application comprises three sets of primer probe combinations; the forward and reverse primers and the probes of the first set of primer probe combination are sequences shown in Seq ID No.1-3 in sequence; the forward and reverse primers and probes of the second set of primer probe combination are sequences shown in Seq ID No.4-6 in sequence; the forward and reverse primers and the probes of the third set of primer probe combination are sequences shown in Seq ID No.7-9 in sequence; the 5 'ends of the three probes are modified with different fluorescent groups, and the 3' ends are modified with fluorescent quenching groups. The reagent provided by the application can identify different gene-deleted vaccines in the same reaction by adopting a combination of three sets of primer probes, provides a specific and sensitive detection reagent for the rapid identification of the African swine fever vaccine, provides a powerful scientific tool for preventing the spread of the African swine fever virus, and has important significance for the effective prevention and control of the epidemic situation of the African swine fever.

Description

Reagent for identifying African swine fever gene deletion vaccine, detection method and application

Technical Field

The application relates to the field of detection of African swine fever vaccines, in particular to a reagent for identifying African swine fever gene deletion vaccines, a detection method and application.

Background

African Swine Fever (ASF) is an acute, virulent and highly-contact animal infectious disease of domestic pigs and wild pigs caused by African Swine Fever Virus (ASFV), and pigs of all varieties and ages can be infected, and the morbidity and mortality can reach 100 percent at most. Although African swine fever is not a disease shared by human and livestock, the disease has great harm to the pig industry, once epidemic situation occurs, the disease is difficult to eradicate in a short time, and the disease is a disease which is mainly prevented by the pig industry in the world. The world animal health Organization (OIE) ranks African swine fever as a legal report epidemic disease, and China ranks it as a type of animal epidemic disease.

The African swine fever occurs for the first time in 1921, so that the African swine fever has been in history for nearly one hundred years, and 62 countries occur in succession in the world. In 8 months in 2018, the African swine fever epidemic situation occurs for the first time in Liaoning province in China, and as 4 days in 4 months in 2019, 118 African swine fever epidemic situations occur sequentially in 29 provinces in China, wherein 115 epidemic situations are epidemic situations of live pigs, 3 epidemic situations of wild pigs, and nearly one million live pigs are killed in an accumulated mode. In 2018, the epidemic situation of the African swine fever is more active all over the world, 6500 outbreaks occur in 25 countries such as Russia, Romaniia and Poland all over the world, and a part of the countries present a big outbreak and a big epidemic situation. In 62 countries with epidemic situation of African swine fever, only 13 countries eradicate the epidemic situation, the eradication time is 5 to 36 years, and a great deal of manpower, material resources and financial resources are consumed. Russia has not yet been effectively controlled since the first outbreak of African swine fever in 2007 to date by over 1000. China is a big pig-raising country and a big pork-consuming country, the annual consumption of pork exceeds 5000 million tons, and the prevention and control effect of the epidemic situation of the African swine fever is crucial to the harmonious and stable society of China and the development of the pig-raising industry.

Because the traditional breeding structure in China, such as low breeding scale degree, high live pig transportation frequency and large range, combines the successful experience of the existing animal epidemic disease prevention and control, and the vaccine is the most effective and economic method for preventing and controlling the epidemic situation of the African swine fever. However, as of today, there is no approved commercial available African swine fever vaccine in countries around the world, including China, and the farms/households expect safe and effective vaccines to be on the market as soon as possible.

The molecular biology method is adopted to knock out a virus functional gene, a virus virulence gene or an immunosuppression gene, so that the virus virulence can be reduced or the immune response of an organism to the virus can be increased, and the development of the gene deletion attenuated live vaccine which has better safety and higher efficacy compared with the traditional attenuated vaccine is one of important directions for researching the African swine fever vaccine. Foreign researches on African swine fever gene deletion viruses are carried out, for example, O' DonnellV and the like report that attenuated viruses deleting multigene families MGF360 and MGF505 can cause the protection of organisms on the virus attack of parent strains. Research shows that the vaccine modified by the gene can provide wider immune protection, for example, BA71(I type separated strain) which is reported by JENSON JS and the like and lacks CD2v/EP402R gene can not only generate immune protection to parent strain, but also generate protection to E75 strain of the same genus I and Georgia07 strain from type II. Meanwhile, researchers in China also have carried out researches on MGF360-505R gene deletion and gene deletion viruses with CD2v and MGF360-505R combined deletion.

Although, at present, no African swine fever vaccine approved for sale at home and abroad exists; however, current studies show that the african swine fever gene deletion vaccine is the most likely and promising african swine fever vaccine. Therefore, in the foreseeable future, how to effectively distinguish the African swine fever gene deletion vaccine from the wild strain infected with the disease will interfere with the prevention and control of the African swine fever seriously. Therefore, a detection method and a reagent capable of identifying the African swine fever gene deletion vaccine and the wild strain are urgently needed, technical support is provided for the subsequent popularization and application of the African swine fever gene deletion vaccine, and the detection method and the reagent have important significance for the effective prevention and control of the African swine fever.

Disclosure of Invention

The application aims to provide a new reagent for identifying African swine fever gene deletion vaccine, a detection method and application.

The following technical scheme is adopted in the application:

one aspect of the application discloses a reagent for identifying African swine fever gene deletion vaccine, which comprises three sets of primer probe combinations; in the first set of primer probe combination, the forward primer is a sequence shown by Seq ID No.1, the reverse primer is a sequence shown by Seq ID No.2, and the probe is a sequence shown by Seq ID No. 3; in the second set of primer probe combination, the forward primer is a sequence shown by Seq ID No.4, the reverse primer is a sequence shown by Seq ID No.5, and the probe is a sequence shown by Seq ID No. 6; in the third set of primer and probe combination, the forward primer is a sequence shown by Seq ID No.7, the reverse primer is a sequence shown by Seq ID No.8, and the probe is a sequence shown by Seq ID No. 9;

Seq ID No.1：5’-AACGCGTTCGCTTTTCG-3’

Seq ID No.2：5’-CATCGTGGTGGTTATTGTTGGT-3’

Seq ID No.3：5’-ACGTGTCCATAAAACGCAGGTGACCC-3’

Seq ID No.4：5’-GGGTTCGGATACAGGCGTTA-3’

Seq ID No.5：5’-CGTGTTCCTGCCGTGTATCTAA-3’

Seq ID No.6：5’-CCTCCCAGTTCCGCACACAGCC-3’

Seq ID No.7：5’-GAAGAACAATGTCAGCATGATGA-3’

Seq ID No.8：5’-ACTGTAAGGCTTAGGAAGTAATGGTT-3’

Seq ID No.9：5’-ACCACTTCCATACATGAACCATCTCCCA-3’

wherein, the probes of the first set of primer-probe combination with the sequence shown in Seq ID No.3, the probes of the second set of primer-probe combination with the sequence shown in Seq ID No.6 and the probes of the third set of primer-probe combination with the sequence shown in Seq ID No.9, the 5 'ends of the three probes are modified with different fluorescent groups, and the 3' ends are modified with fluorescent quenching groups.

In the reagent of the present application, the first set of primer-probe combination is a primer and a probe for specifically detecting the African swine fever virus B646L gene, the second set of primer-probe combination is a primer and a probe for specifically detecting the MGF _360-14L gene, and the third set of primer-probe combination is a primer and a probe for specifically detecting the CD2v (EP402R) gene. The key point of the application is that a primer and probe combination which can be used for triple real-time fluorescent PCR detection is designed according to three genes; wherein, the modification of different fluorescent groups at the 5' ends of the three probes is to simultaneously detect the three genes. Therefore, when the reagent is used for identifying the African swine fever gene deletion vaccine, the African swine fever vaccine with MGF _360-14L gene and/or CD2v (EP402R) gene deletion can be effectively identified; for example, for wild strains, the three primer probe sets all have amplification signals; for the African swine fever vaccine with MGF-360-14L gene deletion, only the first primer probe combination and the third primer probe combination have amplification signals; in the case of the African swine fever vaccine with combined deletion of MGF _360-14L gene and CD2v (EP402R) gene, only the first set of primer probes combined with the amplification signal.

It is understood that the primer and probe combinations of the present application are designed for triple real-time fluorescent PCR detection; however, in practical applications, each primer probe set can be used alone or in combination of two primer probe sets, and the functions of the primer probe sets are different. For example, the first set of primer probe combination can be used for detecting the African swine fever virus nucleic acid independently, and can be detected whether a vaccine strain or a wild strain; the second set of primer probe combination can be independently used for detecting the African swine fever virus wild strain; the third set of primer and probe combination can also be independently used for detecting the African swine fever virus wild strain. In addition, the first set of primer probe combination and the second set of primer probe combination are used together, and the combination can be used for identifying the African swine fever vaccine with MGF _360-14L gene deletion. The first set of primer probe combination and the third set of primer probe combination are used together, and can be used for identifying the African swine fever vaccine with CD2v (EP402R) gene deletion; however, in general, there is no african swine fever vaccine with CD2v (EP402R) gene deletion alone; thus, a first primer probe set alone is not generally used in combination with a third primer probe set.

Preferably, in the probe of the first set of primer probe combination with the sequence shown in Seq ID No.3, the 5 'end is modified with 6-FAM, and the 3' end is modified with BHQ 1; in the probes of the second primer probe combination with the sequence shown in Seq ID No.6, Cy5 is modified at the 5 'end, and BHQ2 is modified at the 3' end; in the probe of the third set of primer-probe combination having the sequence shown in Seq ID No.9, the 5 '-end is modified with VIC, and the 3' -end is modified with BHQ 1.

It should be noted that the key point of the present application is to design a combined primer and a probe for triple real-time fluorescent PCR detection, it can be understood that, in principle, only different fluorophores need to be modified at the 5' ends of the three probes, and the 6-FAM, Cy5 and VIC fluorophores are only specifically used in one implementation manner of the present application, and it is not excluded that other fluorophores capable of being combined by three may also be used. As for the fluorescence quenching group modified at the 3' end, the fluorescence quenching group is not limited to BHQ1 and BHQ2 as long as the fluorescence quenching group can be compatible with the corresponding fluorescent group.

On the other hand, the application discloses application of the reagent in preparation of a kit or a gene chip for detecting the African swine fever gene deletion vaccine.

It can be understood that the reagent of the application can specifically detect the African swine fever vaccine with MGF _360-14L gene deletion and the African swine fever vaccine with MGF _360-14L gene and CD2v (EP402R) gene combined deletion, therefore, a corresponding African swine fever vaccine detection kit or gene chip can be prepared. As for the gene chip, it may be a liquid phase chip or a solid phase chip, for example, probes or their reverse complements may be immobilized on a chip substrate or microspheres, and then whether MGF-360-14L gene is deleted or MGF-360-14L gene and CD2v (EP402R) gene are deleted in combination may be determined by detecting PCR amplification products. It can be understood that if the detection is carried out by using a gene chip, the three probes do not need to be marked with a fluorescent group and a fluorescence quenching group.

In another aspect, the application discloses a kit for identifying an African swine fever gene deletion vaccine, and the kit contains the reagent of the application.

Preferably, the kit of the present application further comprises reagents for real-time fluorescent PCR.

It should be noted that the reagent of the present application is made into a kit for convenience, and therefore, in principle, the kit may contain all reagents to be used, such as a reagent for real-time fluorescence PCR; of course, in order to reduce the amount of the kit, only the reagent of the present application may be included, and other reagents required to be used may be directly purchased from real-time fluorescent PCR reagents conventionally used in laboratories.

On the other hand, the application discloses a detection method of the African swine fever gene deletion vaccine, which comprises the steps of carrying out real-time fluorescence PCR detection on a sample to be detected by adopting the reagent or the kit of the application, and judging whether the African swine fever gene deletion vaccine is obtained according to the detection result.

The method for detecting the African swine fever gene deletion vaccine mainly aims at detecting non-diagnostic treatment of the African swine fever vaccine aiming at an ex vivo sample, for example, detection in the process of vaccine research, detection of entry and exit products and the like, and does not relate to disease diagnosis or treatment detection of living pigs.

Preferably, the real-time fluorescent PCR detection is triple real-time fluorescent PCR detection, and the specific method for judging whether the African swine fever gene deletion vaccine is detected is that the first set of primer probe combination, the second set of primer probe combination and the third set of primer probe combination all have amplification signals, and the vaccine is judged to be a wild strain; and if the first set of primer probe combination has an amplification signal and at least one of the second set of primer probe combination and the third set of primer probe combination has no amplification signal, the African swine fever gene deletion vaccine is judged. More specifically, the first set of primer probe combination and the third set of primer probe combination have amplification signals, and the second set of primer probe combination has no amplification signals, so that the African swine fever vaccine with MGF-360-14L gene deletion is judged; only the first set of primer probe combination has amplification signals, and the second set of primer probe combination and the third set of primer probe combination have no amplification signals, the African swine fever vaccine with combined deletion of MGF _360-14L gene and CD2v (EP402R) gene is judged.

It should be noted that the first set of primer probe combination is a gene possessed by all African swine fever viruses, and is possessed by both wild strains and vaccine strains, so if the first set of primer probe combination does not amplify signals, the detection result may be problematic, or the detection object is not infected.

The beneficial effect of this application lies in:

according to the reagent for identifying the African swine fever gene deletion vaccine, the African swine fever vaccine with MGF _360-14L gene deletion and the African swine fever vaccine with MGF _360-14L gene and CD2v (EP402R) gene combined deletion can be identified in the same reaction through specific detection of three sets of primer probe combinations, a specific and sensitive detection reagent is provided for rapid identification and detection of the African swine fever gene deletion vaccine, a foundation is laid for application of the African swine fever gene deletion vaccine, a powerful scientific tool is provided for preventing propagation of African swine fever virus, and the reagent has important significance for effective prevention and control of African swine fever.

Drawings

FIG. 1 shows the results of triple real-time fluorescence PCR specific detection in the examples of the present application;

FIG. 2 shows the triple fluorescence PCR detection results of the nucleic acid of the gene deletion vaccine strain in the examples of the present application;

FIG. 3 shows the result of detecting nucleic acid of a gene deletion vaccine strain by the fluorescence PCR method recommended by OIE in the examples of the present application;

FIG. 4 shows the results of the sensitivity test for the B646L gene in the examples of the present application;

FIG. 5 is a real-time fluorescence PCR standard curve for the B646L gene in the examples of the present application;

FIG. 6 shows the results of the sensitivity test for MGF-360-14L gene in the examples of the present application;

FIG. 7 is a real-time fluorescence PCR standard curve for MGF-360-14L gene in the examples of the present application;

FIG. 8 shows the results of the sensitivity test for CD2v gene in the examples of the present application;

FIG. 9 is a real-time fluorescence PCR standard curve for the CD2v gene in the examples of the present application.

Detailed Description

At present, no African swine fever gene deletion vaccine product exists, however, the present application previously researches the African swine fever vaccine which is possibly used and has MGF _360-14L gene deletion, and the African swine fever vaccine which has MGF _360-14L gene and CD2v (EP402R) gene combined deletion, and proposes triple real-time fluorescent PCR reagents which can identify the two African swine fever gene deletion vaccines, namely three sets of primer probe combinations which are specially designed by the present application.

The reagent for identifying the African swine fever gene deletion vaccine can accurately and effectively identify a wild strain and two African swine fever gene deletion vaccines, avoids the interference of the use of the two African swine fever gene deletion vaccines on the prevention and control of the African swine fever, and provides technical support for the popularization and the application of the African swine fever gene deletion vaccines.

The partial terms referred to in this application are to be construed as follows:

wild strains: refers to a normal, toxic African swine fever virus without gene deletion.

Vaccine strain: refers to a strain which is subjected to gene deletion and can be used as an African swine fever vaccine.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

Materials and methods

1. Plasmid and nucleic acid sample for test

Positive plasmid samples: in this example, three gene fragments, namely the B646L gene, the MGF _360-14L gene and the CD2v (EP402R) gene of African swine fever virus ASFV/CN/HLJ/18 strain, were synthesized, and cloned into a pUC57 vector to prepare positive plasmid samples of this example, which were labeled pUC-B646L and pUC-360-14L, pUC-CD2v, respectively. Three genes were synthesized by Biotechnology engineering (Shanghai) GmbH. The three gene fragments were cloned with reference to the pUC57 vector using instructions, which are not described herein.

Viral nucleic acid sample: the viral nucleic acid samples tested in this example include inactivated viral nucleic acids of African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome (PRRSV), porcine circovirus type II (PCV2), foot-and-mouth disease virus (FMDV), porcine type A Selenecar Virus (SVA), and the like. All virus nucleic acid samples are stored and provided by Shenzhen customs animal and plant inspection and quarantine technology center.

2. Primary reagents and instruments

THUNDERBIRD Probe qPCR Mix, a product of Toyo Boseki; nucleic acid extraction Kit MagMAXXTM-96 Viral RNArelationship Kit (AM1836), product of Thermo corporation; a KingFisher Flex full-automatic magnetic bead extraction and purification system, a product of Thermo company; a table top high speed Centrifuge (Centrifuge5415R), a product of Eppendorf corporation; 7500Fast real-time fluorescence PCR instrument, ABI products.

3. Design and screening of real-time fluorescent PCR primers and probes

In this example, a plurality of specific primers and probes were designed for screening based on the African swine fever virus B646L gene, MGF _360-12L gene, MGF _360-13L gene, MGF _360-14L gene, MGF _505-2R gene and CD2v (EP402R) gene. Specifically, four groups of primers and four corresponding probes are designed for the B646L gene, one group of primers and probes are designed for the MGF _360-12L gene, one group of primers and probes are designed for the MGF _360-13L gene, one group of primers and probes are designed for the MGF _360-14L gene, one group of primers and probes are designed for the MGF _505-2R gene, three groups of primers and three corresponding probes are designed for the CD2v (EP402R) gene, and the reverse primers of the third group of primers are the same as the forward primers of the second group.

Specific sequences of all designed primers and probes are shown in Table 1. All primers and probes of this example were synthesized by Biotechnology (Shanghai) Inc.

TABLE 1 real-time fluorescent PCR primer and Probe sequences

In Table 1, in the probe having the sequence shown in Seq ID No.3, 6-FAM was modified at the 5 '-end and BHQ1 was modified at the 3' -end; in the probe with the sequence shown in Seq ID No.6, Cy5 is modified at the 5 'end, and BHQ2 is modified at the 3' end; in the probe having the sequence shown in Seq ID No.9, VIC was modified at the 5 'end and BHQ1 was modified at the 3' end. In the probe with the sequence shown in Seq ID No.12, Cy5 is modified at the 5 'end, and BHQ2 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.15, Cy5 is modified at the 5 'end, and BHQ2 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.18, Cy5 is modified at the 5 'end, and BHQ2 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.21, 6-FAM is modified at the 5 'end, and BHQ1 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.24, 6-FAM is modified at the 5 'end, and BHQ1 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.27, HEX is modified at the 5 'end, and BHQ1 is modified at the 3' end; in the probe with the sequence shown in Seq ID No.30, 6-FAM is modified at the 5 'end, and BHQ1 is modified at the 3' end; in the probe having the sequence shown in Seq ID No.32, Cy3 was modified at the 5 '-end, and BHQ2 was modified at the 3' -end.

4. Reaction system and reaction conditions

Triple fluorescent PCR amplification was performed using the THUNDERBIRD Probe qPCR Mix kit. And a matrix method is used for carrying out triple fluorescence PCR, and the matching concentration of the primer and the probe and the annealing extension temperature and time are screened so as to obtain the optimal amplification effect. The reaction system and reaction conditions were as follows:

the reaction system is 25 μ L, wherein the Probe qPCRMix is 12.5 μ L, 6 primers (10 μmol/L) are 0.5 μ L respectively, 3 probes (10 μmol/L) are 0.3 μ L respectively, distilled water is 3.6 μ L, and the template is 5 μ L.

The reaction condition is pre-denaturation at 95 ℃ for 40 s; then 40 cycles are entered: 8s at 95 ℃ and 45s at 58 ℃; three fluorescence signals, FAM, Cy5 and VIC, were collected at 58 ℃.

Wherein, the 6 primers refer to forward primers and reverse primers of three sets of primer probe combinations; the 3 probes refer to probes in the three sets of primer probe combinations; in the three sets of primer probe combinations, one set of primer probe combination is a specific primer probe of the B646L gene, one set of primer probe combination is a specific primer probe of the CD2v (EP402R) gene, and the other set is a specific primer probe of the MGF _360-12L gene, the MGF _360-13L gene, the MGF _360-14L gene or the MGF _505-2R gene.

5. Specificity test

The screened primer and probe combination is adopted, and according to '4 reaction systems and reaction conditions', inactivated virus nucleic acids such as ASFV, CSFV, PRRSV, PCV2, FMDV, SVA and the like are respectively used as templates to carry out triple real-time fluorescence PCR detection, and a negative control and a water blank control confirmed by the fluorescence PCR method recommended by OIE are set.

Meanwhile, the specificity of the same sample is verified by adopting a fluorescence PCR method recommended by OIE.

6. Detection of African swine fever CD2v and MGF360-505R gene combined deletion vaccine strain nucleic acid

In this example, two African swine fever CD2v and MGF360-505R genes were combined to delete the nucleic acid of the vaccine strain. The two nucleic acids of the vaccine strain with the deletion of the CD2v and MGF360-505R genes are presented by Beijing veterinary research institute of Chinese academy of agricultural sciences.

And (3) detecting two copies of African swine fever CD2v and MGF360-505R gene combined deletion vaccine strain nucleic acid by adopting the screened primer and probe combination according to a reaction system and reaction conditions, wherein each copy is made into two parallel samples, and a negative control confirmed by an OIE recommended fluorescent PCR method, a water blank control and an ASFV inactivated virus nucleic acid positive control are arranged.

Meanwhile, the same samples were tested and verified by the fluorescence PCR method recommended by OIE.

7. Sensitivity test and standard curve drawing

The concentrations of the synthesized plasmids were all 8. mu.g/mL, and the copy numbers of three plasmids, pUC-B646L and pUC-360-14L, pUC-CD2v, were calculated according to the DNA copy number calculation formula to be 1.572X 10¹²copies/mL、1.934×10¹²copies/mL、1.929×10¹²copies/mL. Respectively diluting plasmid standard substances pUC-B646L and pUC-360-14L, pUC-CD2v by 10-fold gradient, and diluting with 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9The plasmid of the dilution is used as a standard template, each dilution is repeated for three times, the screened primer and probe combination is adopted to carry out triple real-time fluorescence PCR detection for sensitivity analysis, and ABI 7500software V2.0.6 software is adopted to draw a standard curve.

Therein, 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9Dilution, in the case of pUC-B646L, i.e.corresponding copy numbers in the order 1.572X 10⁹copies/mL、1.572×10⁸copies/mL、1.572×10⁷copies/mL、1.572×10⁶copies/mL、1.572×10⁵copies/mL、1.572×10⁴copies/mL、1.572×10³copies/mL. pUC-360-14L and pUC-CD2v were similar.

8. Repeatability test

Each using 10^-4、10^-6Plasmid standard products pUC-B646L and pUC-360-14L, pUC-CD2v with diluted concentrations are used as templates, screened primer and probe combinations are adopted, triple fluorescence PCR tests are carried out according to '4 reaction systems and reaction conditions', and the stability of the PCR products is analyzed by repeated detection for three times.

9. Detection of samples

And performing triple fluorescence PCR detection on 230 parts of frozen pig products, 45 parts of pork products, 480 parts of pig anticoagulation blood and 250 parts of pig lymph nodes according to '4. reaction system and reaction conditions' by adopting the screened primer and probe combination. All samples are provided and preserved by Shenzhen customs movement center.

The same samples were also tested using real-time fluorescent PCR as recommended by OIE.

Second, results and analysis

1. Primer and probe screening

After screening, three primer probe sets were finally screened. The first set of primer probe combination is used for specifically detecting the B646L gene, the second set of primer probe combination is used for specifically detecting the MGF _360-14L gene, and the third set of primer probe combination is used for specifically detecting the CD2v (EP402R) gene. Specifically, a first set of primer-probe combination is composed of a forward primer ASF-P72-F3 shown in Seq ID No.1, a reverse primer ASF-P72-R3 shown in Seq ID No.2 and a probe ASF-P72-P3 shown in Seq ID No.3, and is used for specific detection of a B646L gene. The forward primer ASF360-14L-F shown in Seq ID No.4, the reverse primer ASF360-14L-R shown in Seq ID No.5 and the probe ASF360-14L-P shown in Seq ID No.6 form a second set of primer probe combination for specific detection of MGF _360-14L gene. The forward primer ASF-CD2V-F2 shown in Seq ID No.7, the reverse primer ASF-CD2V-R2 shown in Seq ID No.8 and the probe ASF-CD2V-P2 shown in Seq ID No.9 form a third set of primer-probe combination for specific detection of the CD2v gene.

The three sets of primer probe combination can be well suitable for triple real-time fluorescent PCR amplification, and can effectively detect corresponding targets simultaneously.

2. Results of specificity test

The first set of primer probe combination from Seq ID No.1 to Seq ID No.3, the second set of primer probe combination from Seq ID No.4 to Seq ID No.6 and the third set of primer probe combination from Seq ID No.7 to Seq ID No.9 are adopted to carry out specificity detection on inactivated antigen nucleic acid of ASFV, CSFV, PRRSV, PCV2, FMDV and SVA, and the results are shown in FIG. 1. In fig. 1, 1a, 1b, and 1c are amplification curves of three sets of primer probe combinations against ASFV inactivated virus nucleic acid, 1a is an amplification curve of a first set of primer probe combination, 1b is an amplification curve of a second set of primer probe combination, and 1c is an amplification curve of a third set of primer probe combination, respectively; the remaining curves 2-8 are the amplification curves for inactivated viral nucleic acids of CSFV, PRRSV, PCV2, FMDV, and SVA, respectively, as well as negative control, water blank control.

As can be seen from the results in FIG. 1, only ASFV inactivated virus nucleic acid shows three fluorescence curves, wherein 1a is FAM fluorescence curve, 1b is Cy5 fluorescence curve, and 1c is VIC fluorescence curve, the result is positive, which indicates that the template is wild strain virus nucleic acid; while other pathogenic nucleic acids and blank water controls have no fluorescent signal and are negative. Therefore, the three sets of primer probe combination can carry out specificity detection on African swine fever B646L gene, MGF _360-14L gene and CD2v gene, has no cross reaction with CSFV, PRRSV, PCV2, FMDV and SVA inactivated virus nucleic acid, and has good specificity. The detection result is consistent with the fluorescence PCR method recommended by OIE.

3. Detection result of African swine fever CD2v and MGF360-505R gene combined deletion vaccine strain nucleic acid

The detection results of two African swine fever gene deletion vaccines are shown in figure 2. In FIG. 2, sample 1 and sample 2 represent the amplification curves of two African swine fever gene deletion vaccines, 5a, 5b, and 5c represent the amplification curves of three primer probe sets for positive control, and 3 and 4 represent the amplification curves of negative control and water blank control, respectively.

The results in FIG. 2 show that the results for the positive control, negative control and water blank are all normal, indicating that triple real-time fluorescent PCR is proceeding normally; both sample 1 and sample 2 have only FAM fluorescence curves, which indicate that the B646L gene is amplified, but the MGF _360-14L gene and the CD2v gene have no amplification curves, which is consistent with the gene deletion type of the African swine fever vaccine.

The detection results of two African swine fever gene deletion vaccines, positive control, negative control and water blank control by adopting the fluorescence PCR method recommended by OIE are shown in figure 3, a sample 1 and a sample 2 are amplification curves of the two African swine fever gene deletion vaccines, 5 is an amplification curve of the positive control, and 3 and 4 are amplification curves of the negative control and the water blank control. The results in FIG. 3 show that the amplification curves of both African swine fever gene deletion vaccine and the positive control, but no amplification signals of the negative control and the water blank control; the fluorescent PCR method recommended by OIE can accurately detect the African swine fever virus, but cannot distinguish vaccine strains from wild strains.

Therefore, the three sets of primer probe combinations and the triple real-time fluorescent PCR can be used for identifying the African swine fever gene deletion vaccine strain and the wild strain.

4. Sensitivity test and fluorescence PCR standard curve

(1) B646L gene

Diluting 10 times of gradient^-3～10^-9The plasmid standard pUC-B646L (SEQ ID NO: 1.572X 10) in this order⁹copies/mL～1.572×10³copies/mL, triple real-time fluorescent PCR detection, three replicates per dilution, resulting in an amplification curve as shown in FIG. 4 and a standard curve as shown in FIG. 5.

The results in FIG. 4 show that the triple real-time fluorescent PCR of this example was detected at least 10^-9Dilution, corresponding to a concentration of 1.572X 10³copies/mL. The detection limit is comparable to the fluorescent PCR method recommended by OIE. The Slope of a standard curve Slope drawn by ABI 7500software V2.0.6 software is-3.542, the intercept Y-Inter is 45.24, and the correlation coefficient R²0.993, a good linear relationship between the dilutions, as shown in fig. 5, gives the standard curve equation: y is-3.542 x + 45.24. When the detected sample is quantified, the Ct value of the sample can be substituted into the equation, and the copy number of the sample can be calculated.

(2) pUC-360-14L Gene

Diluting 10 times of gradient^-3～10^-9The plasmid standard pUC-360-14L (plasmid DNA) of (1), the concentration being 1.934X 10 in order⁹copies/mL～1.934×10³copies/mL, triple real-time fluorescent PCR detection, three replicates per dilution, resulting in an amplification curve as shown in FIG. 6 and a standard curve as shown in FIG. 7.

The results in FIG. 6 show that the triple real-time fluorescent PCR of this example was detected at least 10^-9Dilution, corresponding to a concentration of 1.934X 10³copies/mL. The standard curve Slope Slope drawn by ABI 7500software V2.0.6 software is-3.037, the Intercept Intercept is 42.506, and the correlation coefficient R²0.997, a good linear relationship between the dilutions, as shown in FIG. 7, gives the standard curve equation: y-3.037 x + 42.506. When the detected sample is quantified, the Ct value of the sample can be substituted into the equation, and the copy number of the sample can be calculated.

(3) pUC-CD2v gene

Diluting 10 times of gradient^-3～10^-9The plasmid standard pUC-360-14L (in order of concentration) was 1.929X 10⁹copies/mL～1.929×10³copies/mL, triple real-time fluorescent PCR detection was performed, with three replicates per dilution, resulting in an amplification curve as shown in FIG. 8 and a standard curve as shown in FIG. 9.

The results in FIG. 8 show that the triple real-time fluorescent PCR of this example was minimally detectable at 10^-9Dilution, corresponding concentration 1.929X 10³copies/mL. The standard curve Slope Slope drawn by ABI 7500software V2.0.6 is-3.325, the Intercept Intercept is 45.507, and the correlation coefficient R²0.998, a good linear relationship between the dilutions, as shown in fig. 9, gives the standard curve equation: y-3.325 x + 45.507. When the detected sample is quantified, the Ct value of the sample can be substituted into the equation, and the copy number of the sample can be calculated.

5. Results of the repeatability test

Each using 10^-4、10^-6Plasmid standard products pUC-B646L and pUC-360-14L, pUC-CD2v with diluted concentrations are used as templates, triple fluorescence PCR tests are carried out according to the 4. reaction system and reaction conditions, and the stability of the plasmid standard products is analyzed by repeating the detection three times. The coefficient of variation within the group (C.V.) and the coefficient of variation between the groups (C.V.) were both calculated from the Ct values of the test results to be less than 2%, indicating that the three sets of primer probe combinations and the triple real-time fluorescent PCR of this example are highly reproducible.

6. Detection of samples

The sample detection result shows that by adopting the three sets of primer probe combinations and the triple real-time fluorescent PCR, no amplification signal appears in 230 parts of frozen pig products, 45 parts of pork products and 480 parts of anticoagulation pig, and the detection result is negative; in 250 pig lymph nodes, 18 samples show three typical fluorescence amplification curves, and 232 samples show no amplification signals. The detection result is the same as the real-time fluorescent PCR method detection result recommended by OIE.

The detection method and the detection reagent for identifying the African swine fever gene deletion vaccine strain and the wild strain have the advantages that the detection time is only 50min, and the same reaction can accurately identify and detect the African swine fever gene deletion vaccine strain and the wild strain.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shenzhen customs animal and plant inspection and quarantine technical center

Wudingxian county animal epidemic disease prevention control center

<120> reagent for identifying African swine fever gene deletion vaccine, detection method and application

<130>19I29370

<160>32

<170>PatentIn version 3.3

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catcgtggtg gttattgttg gt 22

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acgtgtccat aaaacgcagg tgaccc 26

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gggttcggat acaggcgtta 20

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cgtgttcctg ccgtgtatct aa 22

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Claims

1. A reagent for identifying an African swine fever gene deletion vaccine is characterized in that: the reagent comprises three sets of primer probe combinations;

in the first set of primer probe combination, the forward primer is a sequence shown by Seq ID No.1, the reverse primer is a sequence shown by Seq ID No.2, and the probe is a sequence shown by Seq ID No. 3;

in the second set of primer probe combination, the forward primer is a sequence shown by Seq ID No.4, the reverse primer is a sequence shown by Seq ID No.5, and the probe is a sequence shown by Seq ID No. 6;

in the third set of primer and probe combination, the forward primer is a sequence shown by Seq ID No.7, the reverse primer is a sequence shown by Seq ID No.8, and the probe is a sequence shown by Seq ID No. 9;

Seq ID No.1：5’-AACGCGTTCGCTTTTCG-3’

Seq ID No.2：5’-CATCGTGGTGGTTATTGTTGGT-3’

Seq ID No.3：5’-ACGTGTCCATAAAACGCAGGTGACCC-3’

Seq ID No.4：5’-GGGTTCGGATACAGGCGTTA-3’

Seq ID No.5：5’-CGTGTTCCTGCCGTGTATCTAA-3’

Seq ID No.6：5’-CCTCCCAGTTCCGCACACAGCC-3’

Seq ID No.7：5’-GAAGAACAATGTCAGCATGATGA-3’

Seq ID No.8：5’-ACTGTAAGGCTTAGGAAGTAATGGTT-3’

Seq ID No.9：5’-ACCACTTCCATACATGAACCATCTCCCA-3’

2. The reagent according to claim 1, characterized in that: in the probes of the first set of primer probe combination with the sequence shown in Seq ID No.3, 6-FAM is modified at the 5 'end, and BHQ1 is modified at the 3' end; in the probes of the second set of primer probe combination with the sequence shown in Seq ID No.6, the 5 'end is modified with Cy5, and the 3' end is modified with BHQ 2; in the probe of the third set of primer probe combination with the sequence shown in Seq ID No.9, the 5 'end is modified with VIC, and the 3' end is modified with BHQ 1.

3. The use of the reagent according to claim 1 or 2 in the preparation of a kit or gene chip for detecting African swine fever gene deletion vaccine.

4. Use according to claim 3, characterized in that: the African swine fever gene deletion vaccine is an African swine fever vaccine with MGF-360-14L gene and/or CD2v (EP402R) gene deletion.

5. A kit for identifying African swine fever gene deletion vaccine is characterized in that: the kit contains the reagent of claim 1 or 2.

6. The kit of claim 5, wherein: the kit also contains a reagent for real-time fluorescent PCR.

7. A detection method of African swine fever gene deletion vaccine is characterized in that: the method comprises the steps of carrying out real-time fluorescence PCR detection on a sample to be detected by using the reagent of claim 1 or 2 or the kit of claim 5 or 6, and judging whether the vaccine is the African swine fever gene deletion vaccine or not according to the detection result.

8. The detection method according to claim 7, characterized in that: the real-time fluorescent PCR detection is triple real-time fluorescent PCR detection, and the specific method for judging whether the vaccine is the African swine fever gene deletion vaccine is that the first set of primer probe combination, the second set of primer probe combination and the third set of primer probe combination all have amplification signals, and the vaccine is judged to be a wild strain; and if the first set of primer probe combination has an amplification signal and at least one of the second set of primer probe combination and the third set of primer probe combination has no amplification signal, the African swine fever gene deletion vaccine is judged.