CN113293233A - African swine fever virus detection kit - Google Patents

Abstract

The invention relates to an African swine fever virus detection kit, which consists of 20 PCR reaction tubes, 300 mu l of PCR reaction mixture, 2.0g of agarose, 100 mu l of ethidium bromide 10 mu g/mu l, 0.25% bromophenol blue spot-like buffer solution 100 mu l and 5 mu l of Taq enzyme (5U/mu l), wherein the PCR reaction mixture comprises Primer sequences F5'-CTGGCATAGGACGGAGTA-3' and R5'-CGCAACATTCGCATCTAC-3'; the primer sequence contained in the kit provided by the invention is highly conserved, and the kit can detect the mutant African swine fever viruses from different sources and can better cope with the mutation of the African swine fever viruses.

Description

African swine fever virus detection kit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an African swine fever virus detection kit.

Background

African Swine Fever (ASF) is caused by African Swine Fever Virus (ASFV), skilful pigs, porcupines, wild pigs and domestic pigs are susceptible, once the pigs are infected, the main clinical symptoms are high fever, inappetence and skin cyanosis, bleeding of lymph nodes, kidneys, gastrointestinal mucosa and the like can be found after the autopsy, after the pigs are infected by strains with strong toxicity, the fatality rate can reach 100%, although the virus is not reported to infect people, the virus brings great harm to the pig industry after the disease occurs, the sale, trade and the like of animal products are severely limited, so the world animal health organization lists the virus as the animal epidemic disease reported in the famous records of national animal pathogenic microorganisms, and is also listed as a type of animal diseases.

ASFV is icosahedral symmetrical, the diameter of the virus particle is 200nm, the structure is concentric sphere, the most central region is nucleoplasm, the diameter is about 80nm, it is composed of virus genome, enzyme necessary for completing gene early transcription and some DNA binding protein, such as p150, p37, p34, p14 and p14.5, p10, the nucleocapsid composed of some protein is around the nucleoplasm, the capsomer is its basic structural unit, there are 1982 and 2172.

In 1921, ASF was first developed in kenya, the disease occurred in some countries around the sahara desert in the next decades, the first outbreak of the disease occurred in portugal countries outside the african continent in 1957, the ASF of portugal was controlled and eliminated soon after this occurrence, and in 1960, the next outbreak of ASF occurred in areas around this rician, from the beginning of the outbreak to 1995, the spread of ASFV in many areas in the isb peninsula (spain and portugal), and in the last 70 th to the middle and late 80 th century, the spread of ASFV rapidly to other parts of the world, where the impact is greater in other european countries (e.g., italy, france, the netherlands, belgium, etc.) and some countries in the america, and in particular in dominica and brazil. Through root tracing, the swill of airports and ports which pollute the ASFV is fed to healthy pigs, which is the main reason for spreading the ASFV from epidemic areas to epidemic disease-free countries. Once a herd of pigs is infected, the sick pigs and the pork products become new sources of viral infection. Epidemiological data on the ASFV in spain show that the ASF can be cleared if the control measures can be put in place, but the virus persists in the indian italy and in the african continent, especially in the southeast part of africa. The epidemiology and distribution of ASF have changed from the last 90 s to the beginning of this century, beginning to enter other regions, including some western african countries; ASF occurs in Cotdeva in 1996, in Nigeria and in san-Goo in 1997, in 1999 in Kanna, in 2003 in Buckner Fasso, at 2010 at first glance, etc.; while west africa is occurring, the prevalence of ASF has also emerged in island countries such as magas and friesis; in 2007, Gruggia first developed ASF, followed by a northwest epidemic, and then entered the continental Europe. Up to now, ASF has been prevalent in many countries in africa, america, europe and the continental europe border, 8/3 in 2018, the first african swine fever epidemic in china was diagnosed, and before 2018, there was no african swine fever in china. Molecular epidemiological studies have shown that: the African swine fever virus introduced into China belongs to the II type gene, the homology of the gene with the whole genome sequence of strains published by Grugia, Russia and Poland is about 99.95 percent, and generally, the routes of the African swine fever introduced across the national border mainly comprise four types: the method comprises the steps of carrying out international trade and smuggling on live pigs and products thereof, carrying pork and products thereof by international passengers, carrying kitchen residues on international transport means, and migrating wild pigs. The spreading way of the 68 pig epidemic situation which has been proved by China is mainly three: first, live pigs and their products are distributed and transported across regions, accounting for about 19% of all epidemic situations; feeding pigs with kitchen residues, wherein the pig residues account for about 34% of the whole epidemic situation; and thirdly, people and vehicles carry toxic spread, which is the most main mode for spreading the current epidemic situation and accounts for about 46 percent of the whole epidemic situation.

The PCR detection has the characteristics of high detection specificity, strong sensitivity, simple operation, mass detection and the like, is used for detecting the nucleic acid of pathogenic microorganisms in various tissues and body fluids, is more rapid and sensitive compared with other tests, does not need to separate and culture the pathogenic microorganisms, and can carry out mass epidemiological investigation. Steiger et al first designed a pair of PCR primers based on the sequence of the conserved domain of AS FV DNA and detected ASFV nucleic acid in cell culture and infected tissue using the established PCR, although the pair of primers was highly sensitive in detection, false positives could be detected in blood sample detection with the generation of non-specific bands. The invention aims to provide a PCR detection method which has high specificity and can adapt to ASFV variation.

Disclosure of Invention

The invention aims to provide a PCR diagnostic kit suitable for ASFV variation, which has a highly conserved primer sequence, can better cope with the ASFV variation and has high specificity.

The preparation and use processes of the invention are as follows:

1. obtaining an African swine fever sequence from NCBI, obtaining a conserved sequence through NCBI Blast function, and showing a comparison result in figure 1;

2. primers were designed against conserved sequences using Primer Premier 6.0, the designed Primer sequences were: primer F5'-CTGGCATAGGACGGAGTA-3' (Seq ID No.1), R5'-CGCAACATTCGCATCTAC-3' (Seq ID No.2), amplified region 94354-94632, length 279 bp;

3. sample processing

Selecting multiple suspected African swine fever pigs, collecting lymph nodes, kidneys, stomachs, intestines and the like of each sick pig, storing in a refrigerator at-20 ℃, recording, weighing 10g of each sample, shearing, adding 5ml of PBS (the pH value is 7.4), grinding at low temperature by using a tissue homogenizer, repeatedly freezing and thawing for 2 times, grinding again, centrifuging in a low-temperature centrifuge at 5000r/min for 10min, transferring the supernatant into another sterilized centrifuge tube, storing at-20 ℃ for extracting DNA;

4. sample DNA template extraction

Transferring the tissue homogenate to a 1.5ml centrifuge tube, boiling and cracking for 10min, then centrifuging at a low temperature of 10000r/min for 10min, transferring the supernatant into another clean centrifuge tube, and storing at-20 ℃ for later use;

5. kit composition

20 PCR reaction tubes, 300. mu.l PCR reaction mixture, 2.0g agarose, 100. mu.l ethidium bromide 10. mu.g/. mu.l, 0.25% bromophenol blue spot-like buffer 100. mu.l, 5. mu.l Taq enzyme (5U/. mu.l);

PCR amplification

Taking 5 mu l of the sample obtained in the fourth step, adding 15 mu l of PCR reaction mixture and 1U of Taq enzyme, and carrying out PCR reaction program: 5min at 95 ℃, 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 35 cycles; and (3) if the sample has a fluorescence band with a preset size at 72 ℃ for 10min, the sample is positive.

The invention has the beneficial effects that:

the primer sequence contained in the African swine fever virus detection kit provided by the invention is highly conserved, and the African swine fever virus detection kit can detect the mutant African swine fever viruses from different sources and can better cope with the mutation of the African swine fever viruses.

Drawings

FIG. 1 alignment scheme

FIG. 2 is a diagram showing the results of the specificity test

FIG. 3 detection of different African swine fever viruses

FIG. 4 is a graph showing the results of sensitivity test

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Materials and reagents related to the present example are shown in table 1, and experimental instruments are shown in table 2;

TABLE 1 Experimental materials and reagents

TABLE 2 Experimental instrumentation

AB104-N electronic analytical balance	Mettler-Torloduoshanghai Co Ltd
		PTC-200 type PCR instrument	MJResearch, USA
DYY-6D type nucleic acid electrophoresis apparatus	BEIJING LIUYI INSTRUMENT FACTORY
		GelDoc gel imaging system	Bio-Rad Inc. of USA
Sigma3K-15 high-speed freezing centrifugal machine	Sigma Germany Co
		MilliQIntegral ultrapure water instrument	Milipore corporation, USA

1. Obtaining an African swine fever sequence (sequence number: U18466.2) from NCBI, obtaining a conserved sequence through NCBI Blast function, and obtaining an alignment result shown in figure 1;

2. primers were designed against conserved sequences using Primer Premier 6.0, the designed Primer sequences were: primer F5'-CTGGCATAGGACGGAGTA-3' (Seq ID No.1), R5'-CGCAACATTCGCATCTAC-3' (Seq ID No.2), amplified region 94354-94632, length 279 bp;

3. sample processing

Selecting multiple suspected African swine fever pigs, collecting lymph nodes, kidneys, stomachs, intestines and the like of each sick pig, storing in a refrigerator at-20 ℃, recording, weighing 10g of each sample, shearing, adding 5ml of PBS (the pH value is 7.4), grinding at low temperature by using a tissue homogenizer, repeatedly freezing and thawing for 2 times, grinding again, centrifuging in a low-temperature centrifuge at 5000r/min for 10min, transferring the supernatant into another sterilized centrifuge tube, storing at-20 ℃ for extracting DNA;

4. sample DNA template extraction

Transferring the tissue homogenate to a 1.5ml centrifuge tube, boiling and cracking for 10min, then centrifuging at a low temperature of 10000r/min for 10min, transferring the supernatant into another clean centrifuge tube, and storing at-20 ℃ for later use;

5. kit composition

20 PCR reaction tubes, 300. mu.l PCR reaction mixture, 2.0g agarose, 100. mu.l ethidium bromide 10. mu.g/. mu.l, 0.25% bromophenol blue spot-like buffer 100. mu.l, 5. mu.l Taq enzyme (5U/. mu.l);

PCR amplification

Taking 5 mu l of the sample obtained in the fourth step, adding 15 mu l of PCR reaction mixture and 1U of Taq enzyme, and carrying out PCR reaction program: 5min at 95 ℃, 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 35 cycles; and (3) if the sample has a fluorescence band with a preset size at 72 ℃ for 10min, the sample is positive.

Experiment-kit specificity test

1. Collection and treatment of pathological material

Selecting multiple suspected African swine fever pigs, collecting lymph nodes, kidneys, stomachs, intestines and the like of each sick pig, storing in a refrigerator at-20 ℃, recording, weighing 10g of each sample, shearing, adding 2ml of PBS (the pH value is 7.4), grinding at low temperature by using a tissue homogenizer, repeatedly freezing and thawing for 2 times, grinding again, centrifuging in a low-temperature centrifuge at 5000r/min for 10min, transferring the supernatant into another sterilized centrifuge tube, storing at-20 ℃ for extracting DNA;

2. extraction of genomic DNA

Transferring the tissue homogenate to a 1.5ml centrifuge tube, boiling and cracking for 10min, then centrifuging at a low temperature of 10000r/min for 10min, transferring the supernatant into another clean centrifuge tube, and storing at-20 ℃ for later use;

3. the DNA sample obtained in the second step, and cDNA of Porcine Pseudorabies virus (PRV), Porcine circovirus type 2 (PCV 2), Porcine reproductive and respiratory syndrome virus (PRRS), Porcine Japanese Encephalitis Virus (JEV), Porcine Parvovirus (PPI) are used as templates, PCR is performed by using designed primers, and the result is shown in FIG. 2.

According to the experimental results, the kit can amplify specific bands in the hog cholera virus sample, and does not amplify specific bands in a PCR system with PrV, PCV2, PRRS, JEV and PPI cDNA as templates, which indicates that the kit has good specificity.

Experiment two: test of sensitivity of kit

The ASFV DNA, a standard template, at an initial concentration of 400 ng/. mu.l, was expressed as 1: 10. 1: 30. 1: 50. 1: 70. 1: 90. 1: 100 dilutions, 2. mu.l of template from each dilution were tested with ASFV PCR kit and the results observed and shown in FIG. 3.

The results show that PrV DNA templates diluted by 1% can also be detected with the kit.

Experiment three: detection of capability of coping with African swine fever virus variation

Searching different regions with large phase difference from NCBI, artificially synthesizing related 4Kb DNA sequences (MN 641876.1, MH910496.1, MN336500.2, M77121.1, NC044956.1, AY261365.1, NC044942.1 and LR536725.1) in different time periods, inserting a pGEM-T vector, transiently transfecting enterobacter coli DH 36a, selecting a strain with correct sequencing, carrying out amplification culture, carrying out PCR detection by using the kit, and obtaining an experimental result shown in figure 3.

According to the experimental results, target strips appear, which shows that the kit can well detect different types of African swine fever viruses and has a good antiviral mutation function.

Experiment four: comparison with other methods

95 parts of pig blood are collected, samples are respectively detected by the kit, HA and ELISA methods, and the results are observed and shown in Table 3.

TABLE 3 detection rates of African swine fever viruses by different detection methods

Method	Number of positive test	Positive detection Rate (%)
			PCR	23	24.2
ELISA	14	14.7
			HA	17	17.9

Sequence listing

<110> Guangzhou Bojii Biotechnology Ltd

<120> African swine fever virus detection kit

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctggcatagg acggagta 18

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgcaacattc gcatctac 18

Claims (2)

1. The African swine fever virus detection kit is characterized by comprising the following components: 20 PCR reaction tubes, 300. mu.l PCR reaction mix, 2.0g agarose, 100. mu.l ethidium bromide 10. mu.g/. mu.l, 0.25% bromophenol blue spot-like buffer 100. mu.l, 5. mu.l Taq enzyme (5U/. mu.l), wherein the PCR reaction mix comprises the primer sequences: primer F5'-CTGGCATAGGACGGAGTA-3', R5'-CGCAACATTCGCATCTAC-3';

2. the African swine fever virus detection kit is characterized in that the preparation and use processes of the African swine fever virus detection kit are as follows:

1) obtaining an African swine fever sequence from NCBI, and obtaining a conserved sequence through NCBI Blast function;

2) primers were designed against conserved sequences using Primer Premier 6.0, the designed Primer sequences were: primer F5'-CTGGCATAGGACGGAGTA-3', R5'-CGCAACATTCGCATCTAC-3', amplified region 94354-94632, length 279 bp;

3) sample processing

Selecting multiple suspected African swine fever pigs, collecting lymph nodes, kidneys, stomachs, intestines and the like of each sick pig, storing in a refrigerator at-20 ℃, recording, weighing 10g of each sample, shearing, adding 5ml of PBS (the pH value is 7.4), grinding at low temperature by using a tissue homogenizer, repeatedly freezing and thawing for 2 times, grinding again, centrifuging in a low-temperature centrifuge at 5000r/min for 10min, transferring the supernatant into another sterilized centrifuge tube, storing at-20 ℃ for extracting DNA;

4) sample DNA template extraction

Transferring the tissue homogenate liquid into a 1.5ml centrifuge tube, boiling and cracking for 10min, then centrifuging at a low temperature of 10000r/min for 10min, transferring the supernatant liquid into another clean centrifuge tube, and storing at-20 ℃ for later use;

5) kit composition

20 PCR reaction tubes, 300. mu.l PCR reaction mixture, 2.0g agarose, 100. mu.l ethidium bromide 10. mu.g/. mu.l, 0.25% bromophenol blue spot-like buffer 100. mu.l, 5. mu.l Taq enzyme (5U/. mu.l);

6) PCR amplification

Taking 5 mu l of the sample obtained in the fourth step, adding 15 mu l of PCR reaction mixture and 1U of Taq enzyme, and carrying out PCR reaction program: 5min at 95 ℃, 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 35 cycles; and (3) if the sample has a fluorescence band with a preset size at 72 ℃ for 10min, the sample is positive.

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