CN114480726A - Primer probe set, kit and detection method for African swine fever virus nucleic acid detection - Google Patents

Abstract

The invention discloses a primer probe group for detecting nucleic acid of African swine fever virus, a dual RT-qPCR kit and a detection method, wherein the kit comprises two sets of primer probe groups, one set of primer probe group is used for detecting mRNA and DNA of an ASFV structural protein coding gene P72, the other set of primer probe group is used for detecting mRNA of a high-abundance transcribed gene A151R before replication of the DNA at the early stage of virus infection, the dual detection kit is utilized for carrying out one-step RT-qPCR, the time point that the ASFV infected pigs can detect toxin expulsion is advanced as far as possible, the detection rate of ASFV, particularly ASFV natural low virulent strains is greatly improved, and the most efficient auxiliary pig farm implements an ASF tooth extraction purification strategy.

Description

Primer probe set, kit and detection method for African swine fever virus nucleic acid detection

Technical Field

The invention belongs to the technical field of animal pathogenic molecule diagnosis, and particularly relates to a primer probe set, a kit and a detection method for detecting African swine fever virus nucleic acid.

Background

African Swine Fever (ASF) is an acute, highly contagious and high-lethality infectious disease of pigs caused by African Swine Fever Virus (ASFV), and is clinically characterized by high Fever and reticuloendothelial system hemorrhage. Pigs of all ages are susceptible, the fatality rate of susceptible swinery is even as high as 100%, the harm to the pig industry is great, the pigs are classified as A-type animal epidemic diseases which need to be reported by the world animal health Organization (OIE), and China classifies the pigs as a type of animal epidemic diseases which are important to prevent and control.

The ASFV can infect wild pigs and soft ticks besides domestic pigs, and can be transmitted circularly among the three; together with the presence of recessively infected pigs, and the circulation of ASFV-contaminated live pig products, the spread of the disease is very rapid. After ASFV is discovered in African Kenya for the first time in 1921 (Eustace Montgomery,1921), European grapefruits were introduced in 1957, European and American in 70-80 years (Plowright, Parker et al 1969), and ASF epidemic outbreaks in Grugia, Yameiya and Russia in 2007. The disease has spread almost globally from africa by less than a century. In 2018, after the ASF epidemic situation is reported for the first time in Liaoning province in China, the epidemic situation rapidly spreads to the whole country, which not only brings huge economic loss to the pig raising industry in China, but also has considerable influence on the life of the national people in African swine fever as the largest pork consumption country in the whole world.

At present, no effective treatment method exists for ASF, no safe and efficient vaccine is used for prevention and control in a short time, and the prevention and control still depends on quick and accurate diagnosis and killing. The ASF is difficult to distinguish from swine fever and highly pathogenic porcine reproductive and respiratory syndrome in clinical symptoms, so that the establishment of a rapid and accurate detection method is important for the prevention and control of the disease. At present, enzyme-linked immunosorbent assay (ELISA), colloidal gold test strips and nucleic acid detection methods are used for detecting ASFV. With the rapid development of molecular diagnosis technology, the fluorescence quantitative PCR technology not only has high sensitivity and specificity, but also can realize high-throughput detection of a large number of samples, and becomes the first-choice mainstream technology in the field of molecular diagnosis. At present, various fluorescent quantitative PCR kit products are applied to detection of ASFV in China, and the detection target is basically focused on the main structural protein coding gene VP72 of the ASFV.

The factors influencing the detection effect of the fluorescent quantitative PCR mainly comprise three factors: firstly, the factors of the specimen, the collection part of the specimen, the specimen preservation method, the preservation time and the like all influence the target content during detection; secondly, personnel factors, the technical level of experimenters, the operation habits in the actual operation process and the like have direct influence on the detection result; thirdly, the stability of the equipment, the temperature rise and fall speed, the intensity of the exciting light source, the software algorithm and the like also have influence on the judgment of the detection result. The latter two factors can be artificially controlled, but the first factor is mainly determined by the occurrence and development process of the disease and the state of a host, and is difficult to artificially control. When the trend of strong toxicity weakening of an ASFV epidemic strain begins to appear in China, the course of disease of an infected pig is prolonged, the latency is increased, the viral vector of a sampling part is greatly reduced, the influence on the first key factor of detection is great, and the detection difficulty of the ASFV is higher.

We can divide the virus propagation cycle into a pre-replication phase and a post-replication phase based on the event of replication of its genomic DNA. Before the viral genome is replicated in a large quantity, the quantity of virus-associated DNA in infected cells of a body is small, if the infection process is prolonged and the quantity of viruses at the infection sites is small, even the most important virus structure coding gene can be lower than a detectable threshold value, which is the reason why the detection of ASFV natural low-virulent strains is more difficult. Therefore, it is urgently needed to develop a method and a detection kit for improving the detection sensitivity and specificity of the ASFV natural low virulent strain.

Disclosure of Invention

In order to solve the problem of high difficulty in detection of the ASFV natural low virulent strain, the invention selects an mRNA sequence with high abundance before DNA replication in the early stage of virus infection as a detection target of fluorescence quantitative PCR (polymerase chain reaction), adds one-step reverse transcription in the fluorescence quantitative PCR process, realizes the detection of the ASFV, especially the ASFV natural low virulent strain by one-step RT-qPCR (reverse transcription-quantitative polymerase chain reaction), not only advances the time point that the ASFV infected pig can detect the detoxification as far as possible, and implements the tooth extraction purification strategy of the ASF in an auxiliary pig farm with the maximum efficiency, but also greatly improves the molecular detection sensitivity of the ASFV, especially the ASFV natural low virulent strain even if the ASFV natural low virulent strain is used for detecting the structural protein coding gene of the ASFV virus, and has the advantages of greatly improving the detection sensitivity of the ASFV.

In order to achieve the purpose, the invention provides a primer probe set for detecting nucleic acid of African swine fever virus, which comprises the following two sets of primer probe sets:

a primer probe group: the primer probe set is used for detecting mRNA and DNA of African swine fever virus P72 gene, and the sequence of the P72 gene is SEQ ID NO: 1;

b, primer probe group: a primer probe set for detecting mRNA of African swine fever virus A151R gene, wherein the sequence of the A151R gene is SEQ ID NO: 2.

Preferably, the primer probe set comprises: the upstream primer is SEQ ID NO: 3, and the downstream primer is SEQ ID NO: 4, the probe is SEQ ID NO: 5; the b primer probe group comprises: the upstream primer is SEQ ID NO: 6, and the downstream primer is SEQ ID NO: 7, the probe is SEQ ID NO: 8.

Preferably, the 5 ' end of the probe in the a primer probe set is labeled with a fluorescence reporter FAM, the 3 ' end of the probe in the b primer probe set is labeled with a fluorescence reporter VIC, and the 3 ' end of the probe in the b primer probe set is labeled with a fluorescence quencher BHQ 1.

The invention also provides an African swine fever virus nucleic acid detection kit, which comprises the primer probe set for African swine fever virus nucleic acid detection.

Preferably, the kit further comprises a positive control substance, a negative control substance, a buffer solution, an RNase inhibitor, dNTP, an enzyme mixture and nuclease-free water.

Preferably, the positive control is TE buffer solution of positive plasmid pMD-312151 containing integrated P72 gene sequence and A151R gene sequence, and the content of the positive plasmid pMD-312151 in the TE buffer solution is 1 x 10⁵copies/. mu.L; the negative control was sterile water.

The invention also provides a non-diagnosis-purpose African swine fever virus detection method, which is used for detecting the African swine fever virus by using the African swine fever virus nucleic acid detection kit and comprises the following steps:

A. extracting a template of a sample to be detected;

B. preparing an RT-qPCR reaction system;

C. PCR amplification and detection;

D. and (6) judging the result.

Preferably, the total volume of the RT-qPCR reaction system is 25 μ L, which comprises 13.55 μ L of nuclease-free water, 2.5 μ L of 10 × buffer, 0.2 μ L of 25mM dNTP, 0.25 μ L of 100 × RNase inhibitor, 1 μ L of enzyme mixture, 1.25 μ L of 20-fold final concentration a primer probe set, 1.25 μ L of 20-fold final concentration b primer probe set, and 5 μ L of amplified template.

Preferably, the PCR amplification and detection reaction conditions are: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 2 min; the amplification was performed in cycles of 95 ℃ for 10s and 57 ℃ for 30s, and the cycle was repeated 45 times.

Preferably, the result is determined as follows:

the experiment is satisfied with the conditions: the positive control FAM channel and the VIC channel have typical amplification curves, the Ct value of the FAM channel is less than or equal to 30, and the Ct value of the VIC channel is less than or equal to 30; negative control has no amplification curve and no Ct value;

in the case of the experiment, the results were interpreted according to the following criteria: if the Ct value of the FAM channel is less than or equal to 40 or the Ct value of the VIC channel is less than or equal to 40 and a typical amplification curve appears, the African swine fever virus exists in the sample template to be detected; and if both the FAM channel and the VIC channel have no Ct value and no amplification curve, the African swine fever virus cannot be detected in the sample template to be detected.

It is understood that the sequence given in SEQ ID NO: 1 and/or seq id no: 2 is an ASFV detection target, and those skilled in the art will know that different primers and probes can be designed for the ASFV detection target screened by the present invention, and any primers and probes designed for the ASFV detection target, and any reactants, reaction solutions, and kits containing the primers and probes designed for the ASFV detection target are within the scope of the present invention.

The primer probe set and the kit for detecting the African swine fever virus can be used for detecting ASFV nucleic acid in the following samples, including but not limited to whole blood, serum, plasma, nasal swab, saliva, lymph node and pork tissue.

The invention has the beneficial effects that: the existing ASFV molecular detection target focuses on the main structural protein coding gene of ASFV, and the detection object is only DNA, so that the ASFV natural low virulent strain has high detection difficulty and can not detect ASFV infected pigs as early as possible, different from the above situations, the invention screens out the gene A151R with high recorded abundance before DNA replication in early stage of virus infection besides the ASFV structural protein coding gene, mRNA and DNA of the P72 gene and A151R gene are used as detection targets of fluorescence quantitative PCR, two sets of specific primer probe sets are designed aiming at the targets, a dual detection kit containing the specific primer probe sets of the two detection targets of the P72 gene and the A151R gene is prepared, the dual detection kit is utilized to bring forward the time point that the ASFV infected pigs can detect the detoxification as far as possible through one-step RT-qPCR, and the detection rate of ASFV, especially ASFV natural low virulent strain is greatly improved, and the most efficient auxiliary pig farm implements ASF tooth extraction purification strategy.

Drawings

FIG. 1 is an orthogonal experimental amplification curve for two variables of primer concentration gradient and probe concentration gradient according to the present invention;

FIG. 2 is an amplification curve of an assay for evaluating sensitivity of detection using the kit of the present invention;

FIG. 3 is an amplification curve of an assay for evaluating detection specificity of the kit of the present invention;

FIG. 4-FIG. 8 are amplification curves of the assay reproducibility test evaluated by the kit of the present invention; wherein, FIG. 4 is a first amplification curve, FIG. 5 is a second amplification curve, FIG. 6 is a third amplification curve, FIG. 7 is a fourth amplification curve, and FIG. 8 is a fifth amplification curve;

FIG. 9 is an amplification curve for detecting ASFV by the kit of the present invention.

Detailed Description

In order to more clearly express the present invention, the present invention will be further described below with reference to examples. The test methods used in the following examples are all conventional methods unless otherwise specified.

Example 1: RT-qPCR primer and probe design

Selecting a P72/A151R gene sequence of ASFV isolates in China and the adjacent countries, carrying out homology analysis, and simultaneously taking a common housekeeping gene sequence of a pig as a reference for avoiding non-characteristic combination, designing a primer and a Taqman probe aiming at a conserved region and not combined with the common housekeeping gene sequence of the pig, wherein the sequences are shown as follows:

firstly, the nucleotide sequence of an ASFV detection target P72 gene is as follows:

P72：

ttcttaaaccccgcaaattacttttttttaggtactgtaacgcagcacagctgaaccgttctgaagaagaagaaagttaatagcagatgccgataccacaagatcagccgtagtgatagaccccacgtaatccgtgtcccaactaatataaaattctcttgctctggatacgttaatatgaccactgggttggtattcctcccgtggcttcaaagcaaaggtaatcatcatcgcacccggatcatcgggggttttaatcgcattgcctccgtagtggaagggtatgtaagagctgcagaactttgatggaaatttatcgataagattgataccatgagcagttacggaaatgtttttaataataggtaatgtgatcggatacgtaacggggctaatatcagatatagatgaacatgcgtctggaagagctgtatctctatcctgaaagcttatctctgcgtggtgagtgggctgcataatggcgttaacaacatgtccgaacttgtgccaatctcggtgttgatgaggattttgatcggagatgttccaggtaggttttaatcctataaacatatattcaatgggccatttaagagcagacattagtttttcatcgtggtggttattgttggtgtgggtcacctgcgttttatggacacgtatcagcgaaaagcgaacgcgttttacaaaaaggttgtgtatttcaggggttacaaacaggttattgatgtaaagttcattattcgtgagcgagatttcattaatgactcctgggataaaccatggtttaaagcgtatattgcgtctac(SEQIDNO：1)

a specific primer probe set designed aiming at the nucleotide sequence of the P72 gene of ASFV is as follows:

P72-1F：ATCGGATACGTAACGGGGCT(SEQIDNO：3)

P72-1R：TTATGCAGCCCACTCACCAC(SEQIDNO：4)

P72-1P：5'FAM-TGCGTCTGGAAGAGCTGTATCTCTATCCTGAAAGC-BHQ1-3'(SEQIDNO：5)

secondly, the nucleotide sequence of the ASFV detection target A151R gene is as follows:

A151R：

atgatggcgttgttacacaaagaaaagcttatagagtgcatctatcatgagctagaaaatggcgggacaatattgcttctaacaaaaaatattgttgtgtcagaaatttcatacattggcaatacttataaatattttacctttaatgacaatcatgatctgataagcaaagaagatcttaaaggagcaacatccaaaaacattgctaaaatgatttataattggattataaaaaatcctcaaaataataagatttggagtggtgagccgcgtactcaaatttattttgaaaatgatttatatcatacaaattacaatcataaatgtataaaagatttttggaatgtttcaacttcagtcggtcctcatatctttaatgatcgtagcatttggtgtactaaatgcacatccttttacccatttaccaacattatgtcgcccaatatattccaataa(SEQIDNO：2)

a specific primer probe set designed aiming at the nucleotide sequence of A151R gene of ASFV is as follows:

A151R-5F：TGGCGTTGTTACACAAAGAAAAG(SEQIDNO：6)

A151R-5R：AATGTTTTTGGATGTTGCTCCTTT(SEQIDNO：7)

A151R-5P：5'VIC-CTTCTTTGCTTATCAGATCATGATTGTCAT-BHQ1-3'(SEQIDNO：8)

wherein, FAM and VIC in the gene probe are fluorescence reporter groups, and BHQ1 is a fluorescence quenching group.

The primers and the probes are synthesized by Shanghai Baili George Biotechnology limited.

Example 2: preparation of ASFV RT-qPCR detection kit

1RT-qPCR reaction system and reaction condition optimization

1.1 optimization of the reaction system:

according to the theoretical Tm value of the primer, the preliminary reaction program is: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 2.5 min; amplification cycles of 95 ℃ 10sec, 56 ℃ 40sec (fluorescence signal acquisition) and 45 cycles of

And (3) respectively setting two variables of a primer concentration gradient and a probe concentration gradient. In each reaction system (25. mu.L), 0.0625. mu.L, 0.125. mu.L, and 0.25. mu.L of a probe at a concentration of 20. mu.M were added, respectively; primers were added at a concentration of 20. mu.M in an amount of 0.125. mu.L, 0.25. mu.L, 0.5. mu.L, and 1. mu.L, respectively, in combination as shown in Table 1:

TABLE 1 amounts of primers and probes added

Each group of primer probes has six concentration combinations, and the number of the cross combinations of each concentration combination of the two groups of primer probes is 36. The ASFV co-extracted nucleic acid (DNA and RNA) is used as a detection sample, 2 multiple wells are arranged for each combination for testing, the amplification result graph is shown in figure 1, and the optimal mixture ratio of two groups of primer probes is that the P72-1F and P72-1R of a primer group are 0.25 mu L respectively, and the probe P72-1P is 0.125 mu L; b primer sets A151R-5F and A151R-5R each 0.25. mu.L, probe A151R-50.065. mu.L. Two groups of primer probe concentrations are respectively prepared into storage solutions with 20 times of final concentration: primer set a, 20. mu.L of primer P72-1F (20. mu.M), 20. mu.L of primer P72-1R (20. mu.M) and 10. mu.L of probe P72-1P (20. mu.M) in 50. mu.L of non-nucleic acid water; b primer Probe set, 54.8. mu.L of nucleic acid-free water plus 20. mu.L of primer A151R-5F (20. mu.M), 20. mu.L of primer A151R-5R (20. mu.M), and 5.2. mu.L of probe A151R-5P (20. mu.M). The optimal reaction system is finally shown in table 2:

TABLE 2 optimal reaction system (Total volume 25. mu.L)

Components	Volume (mu L)
		Nuclease-free water	13.55
10 Xbuffer solution	2.5
		dNTPs (25 mM each)	0.2
RNase inhibitor (100X)	0.25
		Enzyme mixture	1
20-fold concentration primer probe group	1.25
		20-fold concentration b primer probe group	1.25
Amplification template	5

1.2 optimization of the reaction sequence

ASFV co-extracted nucleic acid is used as an amplification sample and is diluted to medium concentration and low concentration (the template dilution with the CT value of 29-32 is determined to be a medium concentration sample and the template dilution with the CT value of 32-40 is determined to be a low concentration sample by a pre-experiment). Three variables of the pre-denaturation time (2min, 2.5min and 3min), the annealing extension temperature (55 ℃, 56 ℃ and 57 ℃) and the annealing extension time (30sec, 35sec and 40sec) were set, the reaction system optimized in 1.1 was selected, and the medium-concentration and low-concentration samples were amplified respectively, and 3 multiple wells were provided for each combination. According to the best test result, the optimal reaction program of the double fluorescence PCR is determined as follows: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 2 min; the amplification cycles were 95 ℃ for 10sec and 57 ℃ for 30sec (fluorescence signal acquisition), and 45 cycles were repeated.

TABLE 3 optimal reaction procedure

2 preparation of Positive control

2.1 obtaining of recombinant plasmid pMD-72151: the P72/A151R gene sequence of ASFV is synthesized by DNA synthesis technology and then directly connected to pMD18T vector. The synthesis of DAN and the acquisition of recombinant plasmid were carried out by Nanjing Kingsrey Biotech Co., Ltd.

2.2 preparation of recombinant plasmid pMD-72151: dissolving plasmid pMD-72151 provided by Nanjing Kingsrie Biotechnology Limited, taking 1 μ L of transformed Escherichia coli DH5 alpha competent cells, coating the competent cells on an LB plate containing 100 μ g/mL carbenicillin, culturing in a 37 ℃ incubator for 16h, picking 4 single colonies, inoculating an LB broth culture medium, culturing in a 37 ℃ constant temperature shaking table for 12h, extracting plasmids for enzyme digestion and sequencing identification, determining the remaining strains of the unmistakable positive clones, and performing amplification culture to extract plasmids.

2.3 preparing TE buffer solution of positive plasmid pMD-72151, the content of positive plasmid pMD-72151 in the TE buffer solution is 1 x 10⁵copies/. mu.L, TE buffer as positive control.

3 negative control set to sterile, enzyme-free water.

Example 3 ASFV RT-qPCR assay kit sensitivity assessment

The positive quality control plasmid pMD-72151 is subjected to gradient dilution to the content of 50, 5 and 0.5 copies/mu L respectively to be used as amplification samples, the concentration of each sample is taken as 10 multiple wells, the assembled dual fluorescence RT-PCR detection kit is used for detecting the positive quality control plasmid pMD-72151, the CT values of different fluorescence channels corresponding to two targets are shown in a table 4, and the amplification curve is shown in a figure 2:

TABLE 4 evaluation of CT values of samples for sensitivity of the kit of the invention

The detection sensitivity of the kit is 100% when the template concentration is 5 copies/. mu.L, and the detection rate is about 50% when the template concentration is 0.5 copies/. mu.L (5/10).

Example 4 ASFV RT-qPCR detection kit specificity verification

The method comprises the steps of taking nucleic acid extracted from samples of healthy pig whole blood (N1), healthy pork (N2), pig respiratory syndrome virus cell culture fluid (N3), classical swine fever virus cell culture fluid (N4), porcine circovirus type 2 cell culture fluid (N5), porcine pseudorabies virus inactivated vaccine (N6), escherichia coli cell culture fluid (N7), streptococcus suis culture fluid (N8) and mycoplasma hyopneumoniae cell culture fluid (N9) as specific evaluation samples, detecting the specific evaluation samples by using the dual-fluorescence RT-PCR detection kit, wherein each sample is a double tube, and positive control samples are respectively two tubes of an ASFV co-extracted nucleic acid medium concentration (PM) template and a low concentration (PL) template. The Ct values of the different samples are shown in Table 5, and the amplification curves are shown in FIG. 3. The combination graph shows that the ASFV co-extracted nucleic acid sample has fluorescence signals in two detection channels, and the genome of the healthy pig sample or other viruses, bacteria and mycoplasma has no fluorescence signals, which indicates that the kit has good specificity.

TABLE 5 specific evaluation of CT values of samples by the kit of the invention

Example 5 repeatability verification of ASFV RT-qPCR detection kit

The positive quality control plasmid pMD-312151 is diluted in a gradient manner to the content of 1 × 10 respectively⁴、1×10³、1×10²And 1 copies/. mu.L, as an amplification sample, detecting the amplification sample by using the dual-fluorescence RT-PCR detection kit, repeatedly detecting for 5 times, setting a control tube which takes the genome of the pig as the amplification sample for each amplification, and calculating the corresponding coefficient of variation (CV,%) of the two targets under the three template concentrations, wherein the results are shown in Table 6. The results show that the coefficient of variation of the CT value of the detection result of each concentration gradient is less than 2 percent, which indicates that the kit has good repeatability.

TABLE 6 repeatability evaluation of CT value and corresponding CV value (%)

The amplification curves for the reproducibility verification are shown in fig. 4-8, wherein fig. 4 is a first amplification curve, fig. 5 is a second amplification curve, fig. 6 is a third amplification curve, fig. 7 is a fourth amplification curve, and fig. 8 is a fifth amplification curve; FIGS. 4-8 show that the kit of the present invention has good reproducibility.

Example 6 application of ASFV RT-qPCR detection kit

1 sample Pre-treatment

Total nucleic acids of 2 (B1/B2) whole blood samples and 1 nasal swab (S1) of ASFV positive pigs are respectively extracted by a magnetic bead method or an adsorption column method to be used as detection templates. The extraction method specifically refers to the instruction of the commercial kit, and it should be noted that negative extraction control needs to be set for each extraction.

2 RT-qPCR procedure

2.1 preparation of reaction solution

Taking out the components in the kit, and preparing 5 tubes of reaction liquid (25 mu L/reaction) according to the following proportion:

components	Volume (μ L)
		Reaction premix	19
Enzyme mixture	1
		Amplification template	5

2.2 amplification

The PCR instrument is opened, two fluorescence channels of FAM and VIC are selected, and reaction conditions are set according to the following reaction program: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 2 min; the amplification cycles were 95 ℃ for 10sec and 57 ℃ for 30sec (fluorescence signal acquisition), and 45 cycles were repeated.

3 determination of results

The experiment is satisfied with the conditions: the positive control FAM channel and the VIC channel have typical amplification curves, the Ct value of the FAM channel is less than or equal to 30, and the Ct value of the VIC channel is less than or equal to 30; negative controls had no amplification curve and no Ct value. The Ct values of the different samples are shown in Table 7, and the amplification curves are shown in FIG. 9.

TABLE 7 CT values of the test results

As can be seen from table 7 and fig. 9, the positive control and negative control results of the above tests indicate that the experiment is established, and the sample results are interpreted according to the following criteria: if the Ct value of the FAM channel is less than or equal to 40 or the Ct value of the VIC channel is less than or equal to 40 and a typical amplification curve appears, the ASFV exists in the sample to be detected; and if both the FAM channel and the VIC channel have no Ct value and no amplification curve, the ASFV cannot be detected in the sample to be detected. Therefore, it was judged that the samples B1 and S1 contained ASFV, and no ASFV was detected in the sample B2.

The above disclosure is only for a few specific embodiments of the present invention, but the present invention is not limited thereto, and any variations that can be made by those skilled in the art are intended to fall within the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Bode Shenzhong Yuansheng Biotechnology Limited

Primer probe set, kit and detection method for African swine fever virus nucleic acid detection

<130> 20210804

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 809

<212> DNA

<213> African swine fever virus

<400> 1

ttcttaaacc ccgcaaatta ctttttttta ggtactgtaa cgcagcacag ctgaaccgtt 60

ctgaagaaga agaaagttaa tagcagatgc cgataccaca agatcagccg tagtgataga 120

ccccacgtaa tccgtgtccc aactaatata aaattctctt gctctggata cgttaatatg 180

accactgggt tggtattcct cccgtggctt caaagcaaag gtaatcatca tcgcacccgg 240

atcatcgggg gttttaatcg cattgcctcc gtagtggaag ggtatgtaag agctgcagaa 300

ctttgatgga aatttatcga taagattgat accatgagca gttacggaaa tgtttttaat 360

aataggtaat gtgatcggat acgtaacggg gctaatatca gatatagatg aacatgcgtc 420

tggaagagct gtatctctat cctgaaagct tatctctgcg tggtgagtgg gctgcataat 480

ggcgttaaca acatgtccga acttgtgcca atctcggtgt tgatgaggat tttgatcgga 540

gatgttccag gtaggtttta atcctataaa catatattca atgggccatt taagagcaga 600

cattagtttt tcatcgtggt ggttattgtt ggtgtgggtc acctgcgttt tatggacacg 660

tatcagcgaa aagcgaacgc gttttacaaa aaggttgtgt atttcagggg ttacaaacag 720

gttattgatg taaagttcat tattcgtgag cgagatttca ttaatgactc ctgggataaa 780

ccatggttta aagcgtatat tgcgtctac 809

<210> 2

<211> 456

<212> DNA

<213> African swine fever virus

<400> 2

atgatggcgt tgttacacaa agaaaagctt atagagtgca tctatcatga gctagaaaat 60

ggcgggacaa tattgcttct aacaaaaaat attgttgtgt cagaaatttc atacattggc 120

aatacttata aatattttac ctttaatgac aatcatgatc tgataagcaa agaagatctt 180

aaaggagcaa catccaaaaa cattgctaaa atgatttata attggattat aaaaaatcct 240

caaaataata agatttggag tggtgagccg cgtactcaaa tttattttga aaatgattta 300

tatcatacaa attacaatca taaatgtata aaagattttt ggaatgtttc aacttcagtc 360

ggtcctcata tctttaatga tcgtagcatt tggtgtacta aatgcacatc cttttaccca 420

tttaccaaca ttatgtcgcc caatatattc caataa 456

<210> 3

<211> 20

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<213> Artificial sequence

<400> 3

atcggatacg taacggggct 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

ttatgcagcc cactcaccac 20

<210> 5

<211> 35

<212> DNA

<213> Artificial sequence

<400> 5

tgcgtctgga agagctgtat ctctatcctg aaagc 35

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<400> 6

tggcgttgtt acacaaagaa aag 23

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<400> 7

aatgtttttg gatgttgctc cttt 24

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence

<400> 8

cttctttgct tatcagatca tgattgtcat 30

Claims (10)

1. The primer probe set for detecting the nucleic acid of the African swine fever virus is characterized by comprising the following two primer probe sets:

a primer probe group: the primer probe set is used for detecting mRNA and DNA of African swine fever virus P72 gene, and the sequence of the P72 gene is SEQ ID NO: 1;

b, primer probe group: a primer probe set for detecting mRNA of an African swine fever virus A151R gene, wherein the sequence of the A151R gene is SEQ ID NO: 2.

2. The primer probe set for detecting African swine fever virus nucleic acid according to claim 1, wherein the a primer probe set comprises: the upstream primer is SEQ ID NO: 3, and the downstream primer is SEQ ID NO: 4, and the probe is SEQ ID NO: 5; the b primer probe group comprises: the upstream primer is SEQ ID NO: 6, and the downstream primer is SEQ ID NO: 7, and the probe is SEQ ID NO: 8.

3. The primer probe set for detecting African swine fever virus nucleic acid, according to claim 2, wherein the 5 'end of the probe in the a primer probe set is labeled with a fluorescence reporter group FAM, the 3' end of the probe in the b primer probe set is labeled with a fluorescence quencher group BHQ1, the 5 'end of the probe in the b primer probe set is labeled with a fluorescence reporter group VIC, and the 3' end of the probe in the b primer probe set is labeled with a fluorescence quencher group BHQ 1.

4. An African swine fever virus nucleic acid detection kit, which is characterized in that the kit comprises a primer probe set for African swine fever virus nucleic acid detection according to any one of claims 1-3.

5. The African swine fever virus nucleic acid detection kit of claim 4, wherein the kit further comprises a positive control, a negative control, a buffer, an RNase inhibitor, dNTPs, an enzyme mixture, and nuclease-free water.

6. The African swine fever virus nucleic acid detection kit of claim 5, wherein the positive control is TE buffer containing positive plasmid pMD-312151 integrating P72 gene sequence and A151R gene sequence, the positive plasmid pMD-312151 content in the TE buffer is 1 x 10⁵copies/. mu.L; the negative control was sterile water.

7. A non-diagnosis-purpose detection method for African swine fever virus is characterized in that the African swine fever virus nucleic acid detection kit of any one of claims 4-6 is used for detecting the African swine fever virus, and comprises the following steps:

A. extracting a template of a sample to be detected;

B. preparing an RT-qPCR reaction system;

C. PCR amplification and detection;

D. and (6) judging the result.

8. The method of claim 7, wherein the total volume of the RT-qPCR reaction system is 25 μ L, and the RT-qPCR reaction system comprises 13.55 μ L of nuclease-free water, 2.5 μ L of 10 Xbuffer, 0.2 μ L of 25mM dNTP, 0.25 μ L of 100 XRNase inhibitor, 1 μ L of enzyme mixture, 1.25 μ L of 20-fold final concentration a primer probe set, 1.25 μ L of 20-fold final concentration b primer probe set, and 5 μ L of amplification template.

9. The method of claim 7, wherein the PCR amplification and detection reaction conditions are as follows: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 2 min; the amplification was performed in cycles of 95 ℃ for 10s and 57 ℃ for 30s, and the cycle was repeated 45 times.

10. The method of claim 7, wherein the results are determined as follows:

the experiment is satisfied with the conditions: the positive control FAM channel and the VIC channel have typical amplification curves, the Ct value of the FAM channel is less than or equal to 30, and the Ct value of the VIC channel is less than or equal to 30; negative control has no amplification curve and no Ct value;

in the case of the experiment, the results were interpreted according to the following criteria: if the Ct value of the FAM channel is less than or equal to 40 or the Ct value of the VIC channel is less than or equal to 40 and a typical amplification curve appears, the African swine fever virus exists in the sample template to be detected; and if both the FAM channel and the VIC channel have no Ct value and no amplification curve, the African swine fever virus cannot be detected in the sample template to be detected.

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