CN110894556A

CN110894556A - PCR primer, probe, kit and detection method for detecting African swine fever virus infectivity

Info

Publication number: CN110894556A
Application number: CN201911345922.5A
Authority: CN
Inventors: 危宏平; 刘欢; 余军平; 熊进
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-03-20

Abstract

The invention provides a PCR primer, a probe, a kit and a detection method for rapidly detecting African swine fever virus infectivity, wherein the primer sequence is shown as SEQ ID NO.1-2, and the probe sequence is shown as SEQ ID NO. 3. The kit comprises the PCR primer, the probe and the nucleic acid dye which is combined with nucleic acid and has photosensitive property, a sample to be detected is pretreated by the photosensitive nucleic acid dye, the sample which is not pretreated by the photosensitive dye is taken as a reference sample, then the nucleic acid of the sample to be detected and the nucleic acid of the reference sample are extracted, the copy numbers of the African swine fever virus conserved genes of the sample to be detected and the reference sample are respectively detected by a qPCR method, and whether the infectious African swine fever virus exists in the sample to be detected or not is rapidly judged by comparing the copy numbers of the genes of the two. The kit has the advantages of short detection time, strong specificity, reliable result and simple result judgment, is suitable for various prevention and control units of laboratories and basic levels, veterinary stations, large, medium and small farms and the like, and has important significance for judging measures such as sterilization, prevention and control of the African swine fever virus.

Description

PCR primer, probe, kit and detection method for detecting African swine fever virus infectivity

Technical Field

The invention relates to the field of detection of African swine fever viruses, and in particular relates to a PCR primer, a probe, a kit and a detection method for rapidly detecting the infectivity of the African swine fever viruses.

Background

African Swine Fever Virus (ASFV) is a double-stranded DNA virus, a virulent infectious disease virus that infects domestic and wild pigs and causes them to develop acute hemorrhagic fever, and the disease caused by this virus is collectively called African Swine Fever (ASF). ASFV erupts in 2018, 8 months and 3 days in Shenbei new district of Shenyang city in Liaoning province of China for the first time, and is widely spread in China after several months. ASFV has the characteristics of acute infection, high lethality and the like, so that the ASFV has caused great loss to agricultural economy in China and even the world. ASFV was developed since 1921 in kenyan, but no effective vaccine or drug therapy has been developed. Preventing and controlling the spread of the virus, and killing the sick pigs still is the most effective mode. Therefore, the establishment of a rapid African swine fever virus detection method has very important significance for early virus discovery and early virus infection source control.

Because the African swine fever virus wild strain can only be proliferated in the primary pig cells, and the isolation culture time is longer. Therefore, the detection of African swine fever virus is mainly based on nucleic acid PCR detection technology at present. The general process is that a lysis reagent is added into a sample to be detected, then nucleic acid is extracted, and a characteristic sequence of the ASFV virus (mostly detecting B646L (or called p72) gene) is detected by technologies such as PCR amplification or isothermal amplification, so that the method has the advantages of no need of separation, rapidness, sensitivity and the like, and is the most widely used method at present. However, the method can only detect whether the nucleic acid characteristic sequence of the virus exists in the sample, and cannot provide effective information on whether the virus in the sample is complete, infectious and the like. The determination of the African swine fever epidemic situation and whether the virus in the sample is still infectious after being sterilized by using a disinfectant also need to be determined by a classical virus culture and separation method. The isolation culture of the African swine fever virus not only needs to use primary cells and is time-consuming and labor-consuming as mentioned above, but also needs to be carried out in a P3-grade laboratory under the condition of biological safety protection according to the management requirement of Ministry of agriculture on the experimental activity of highly pathogenic animal pathogenic microorganisms, so that the detection of the infectivity of the African swine fever virus by the isolation culture is not feasible for a pig farm or a common veterinary laboratory, and brings great inconvenience to the actual prevention and control of the African swine fever virus, such as the determination of an infection path, the completeness of disinfection and inactivation, the existence of live virus in the treated environment of the pig farm in which the ASF epidemic situation occurs, and the like, and a quick, simple and convenient alternative technology is urgently needed to be developed.

The DNA modification dye, Propidium azide and Propidium bromide (PMA), is a photosensitive dye which is bound with double-stranded DNA with high affinity, and forms stable covalent cross-linking with the bound DNA under strong visible light, thus hindering the amplification of PCR. Meanwhile, the dye has the characteristic of not permeating the intact cell membrane, so that the dye can be used for selectively modifying DNA exposed by the incomplete membrane of a dead cell, and the DNA in a living cell with the intact cell membrane cannot be modified. Viable bacteria numbers were optionally detected by pretreating the bacteria samples with PMA prior to PCR reaction. The method (PMA-qPCR) is well applied to distinguishing and detecting the infectivity of dead bacteria and partial viruses (norovirus, hepatitis A virus, bacteriophage and the like) in food and environmental samples, but has not been reported in the aspects of detecting ASFV infectivity and the like.

Disclosure of Invention

In view of the restriction factors that the existing cell culture separation technology is long in time consumption, ASFV only proliferates in primary pig cells, and the separation culture activity of virus needs to be carried out in a P3-grade laboratory under the condition of biosafety protection, the invention provides a PCR primer, a probe, a kit and a detection method for rapidly detecting the infectivity of African swine fever virus, and the invention has important significance for the actual prevention and control of African swine fever virus, such as determining the infection path, whether the disinfection is thorough, whether the environment of a swine farm with African swine fever epidemic situation has live virus after being treated, eliminating the potential virus transmission path and the like.

In order to realize the purpose, the invention is realized by the following technical scheme:

the first purpose of the invention is to provide a PCR primer and a probe for rapidly detecting the African swine fever virus infectivity, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3. Downloading all nucleic acid sequences (shown as SEQ ID NO. 4) of ASFV B646L (p72) gene from NCBI GenBank, performing homology alignment analysis, and searching conserved sequence of p72 gene. In the p72 conserved nucleic acid sequence region, specific primers and target probes for detecting the conserved sequence of the specific target gene p72 nucleic acid are designed.

The second purpose of the invention is to provide the application of the PCR primer and the probe in detecting the African swine fever virus and preparing the African swine fever virus detection standardized kit.

The third purpose of the invention is to provide a kit for rapidly detecting the infectivity of African swine fever virus, which comprises: 1) the PCR primers and probes, 2) nucleic acid dye combined with nucleic acid and having photosensitive property.

Further, the nucleic acid dye which binds to the nucleic acid and has a photosensitizing property is propidium iodide.

Further, the kit further comprises Taq DNApolymerase, Buffer, dNTP and sterilized water.

The fourth purpose of the invention is to provide a method for rapidly detecting the infectivity of African swine fever virus based on the kit of any one of the above, which comprises the following steps:

(1) dividing a sample to be detected into 2 parts in equal volume; wherein 1 part is pretreated by photosensitive nucleic acid dye, and the other part is added with sterilized water or buffer solution as a comparison sample;

(2) respectively extracting total DNA of two samples to be detected;

(3) respectively taking the two DNAs extracted in the step (2) as templates to carry out qPCR real-time fluorescent quantitative detection; the nucleotide sequence of the primer in the qPCR reaction is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3;

(4) and after the qPCR reaction is finished, rapidly detecting whether the infectious African swine fever virus exists in the sample according to the amplification curve and the cycle threshold of the two samples to be detected in the fluorescent quantitative PCR reaction.

Further, the specific steps of pretreating the sample to be detected by the photosensitive nucleic acid dye PMA comprise the following steps:

1) centrifuging a sample to be detected in an EP tube without DNase and RNase enzymes, and collecting a supernatant;

2) putting 180 mu l of supernatant into an EP tube without DNase and RNase, adding 20 mu l of PMA into the supernatant in a dark place, uniformly mixing, and incubating for 5-15 min in a dark place at room temperature; the initial concentration of the PMA is 100-1000 mu M;

3) the EP tubes were immediately transferred to ice and then PMA-Lite was used^TMAnd (3) photolyzing the sample for 15-30 min by using an LED photolysis instrument, and continuously shaking the sample to ensure that the PMA and the exposed nucleic acid are fully covalently crosslinked.

Further, the qPCR reaction system is: 0.5 mul of Taq DNApolymerase, 2 mul of 10 XBuffer, 1.6 mul of 2.5mM dNTP, 0.4 mul of 20 mul of a primer shown in SEQ ID NO.1, 0.4 mul of a primer shown in 20 mul SEQ ID NO.2, 0.8 mul of a probe shown in 20 mul SEQ ID NO.3, 2 mul of a template and 12.3 mul of sterilized water; the 10 × buffer comprises: 200mM Tris-HCl (pH 8.3), 200mM KCl, 100mM (NH4)2SO4, 20mM MgSO4 and 5 wt% NP 40.

Further, the qPCR reaction conditions are: pre-denaturation at 95 ℃ for 2min, and then circulation for 40 times; the procedure for each cycle was: denaturation at 95 ℃ for 5sec, annealing at 57 ℃ for 30sec, and collecting the FAM fluorescence channel signal, and after the cycle is completed, the reaction is terminated.

Further, the criteria for detecting the presence of infectious African swine fever virus in a sample are: (1) the Ct value of a sample treated by the photosensitive nucleic acid dye is larger than that of a sample not pretreated by the dye, and the sample contains inactivated ASFV; (2) the larger the Δ Ct, the higher the inactivated virus content; (3) the delta Ct is more than or equal to 6 or no typical amplification curve exists in qPCR reaction of the sample group treated by the photosensitive nucleic acid dye, and the Ct is more than 38, which indicates that the virus in the sample is completely inactivated

Therefore, compared with the prior art, the invention has the following beneficial technical effects:

(1) the qPCR detection primers and the probes provided by the invention are designed aiming at a conserved gene sequence p72 of African swine fever virus as a target gene for detection, and experiments prove that the qPCR detection adopting the primers and the probes has strong specificity and anti-interference performance, and the minimum detection limit is 10 copies/mu l.

(2) The kit for detecting ASFV infectivity has the advantages of short detection time, strong specificity, reliable result, simple result judgment and the like. Compared with a method for separating infectious ASFV by cell culture, the detection kit increases the practicability of infectious ASFV detection, can be used for detecting samples such as tissue samples, serum, plasma and feed raw materials in farms, is suitable for prevention and control units at all levels of laboratories and basic levels, veterinary stations, large, medium and small farms and the like, and has important significance for judging measures such as killing and prevention and control of African swine fever viruses.

Drawings

FIG. 1 shows the result of the primer pair and probe specificity test for detecting the target gene p72 in African swine fever virus.

FIG. 2 is an amplification curve for qPCR using primers and probes at different concentrations.

FIG. 3 is a graph of qPCR amplification curves for different probe concentrations with 0.4. mu.M primer concentration.

FIG. 4 is an amplification curve for qPCR at different annealing temperatures.

FIG. 5 is a result of evaluating sensitivity of a target gene p72 for detecting African swine fever virus, wherein the abscissa is pUC57-ASFV-p72 EcoRI single digestion linearized plasmid with different concentration gradients, and the ordinate is corresponding Ct value, FIG. 5A is a qPCR amplification curve of plasmids with different concentrations, and FIG. 5B is a standard curve fitting copy number of plasmids with different concentrations and corresponding Ct value.

FIG. 6 is a schematic diagram of the rapid detection of ASFV infectivity by PMA-qPCR according to the present invention.

FIG. 7 shows the result of optimizing PMA concentration under PMA-qPCR conditions in the present invention.

Detailed Description

The following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents and biomaterials, if not specifically indicated, are commercially available.

Example 1 establishment of Rapid fluorescence quantitative PCR detection method for African Swine fever Virus

Establishment of fast fluorescence quantitative PCR detection method for African swine fever virus

1. Design and screening of PCR primer and probe for detecting ASFV characteristic sequence p72 nucleic acid copy number

According to the sequence of the B646L gene (p72 protein gene) with conserved and stable sequence in the African swine fever virus genome, two specific primers and probes are designed. The specific method comprises the following steps: downloading all nucleic acid sequences (shown as SEQ ID NO. 4) of ASFV B646L (p72) gene from NCBI GenBank, performing homology alignment analysis, and searching conserved sequence of p72 gene. Specific primers and target probes were designed in the region of the conserved nucleic acid sequence of p 72. The primers for detecting the nucleic acid conserved sequence of the specific target gene p72 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is shown as SEQ ID NO. 3.

SEQ ID NO.1 forward primer sequence: 5'-agtggaagggtatgtaag-3'

Reverse primer sequence of SEQ ID NO. 2: 5'-gcagagataagctttcag-3'

Probe sequence of SEQ ID No. 3: FAM-5'-cagctcttccagacgcatgttc-3' -BHQ

2. qPCR reaction system

TABLE 1 qPCR reaction System

Composition of	Volume of
		Taq DNA polymerase	0.5μl
10×buffer	2μl
		dNTP(2.5mM)	1.6μl
ASFV-p72-Forward(20μM)	0.4μl
		ASFV-p72-Reverse(20μM)	0.4μl
ASFV-p72-Probe(20μM)	0.8μl
		Template DNA	2μl
Sterilized water	12.3μl

Wherein 10 × buffer comprises: 200mM Tris-HCl (pH 8.3), 200mM KCl, 100mM (NH)₄)₂SO₄，20mM MgSO₄And 5% NP 40.

3. qPCR reaction conditions

The qPCR reaction conditions were: pre-denaturation at 95 ℃ for 2min, and then cycling 40 times, wherein the program of each cycle comprises denaturation at 95 ℃ for 5sec, annealing at 57 ℃ for 30sec, collecting signals of FAM fluorescence channels, and finishing the reaction after the cycling is finished.

Two, qPCR detection of ASFV-p72 primer and probe specificity test

The implementation selects Streptococcus suis, Streptococcus hemolyticus, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella enteritidis, Escherichia coli O:157, avian influenza virus (H7N8), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and pUC57-ASFV-p72 linearized plasmids as experimental objects. Wherein the ASFV-p72 conserved sequence is synthesized by KAIKORII bioengineering GmbH of Wuhan gold, and constructed into plasmid pUC57-ASFV-p72 (the construction plasmid method is described in Wang, J., Y.Zhang, J.Wang, L.Liu, X.Pang, and W.Yuan.2017.development of a TaqMan-basedreal-time PCR assay for the specific detection of plasmid circovirus 3.JVirol Methods248: 177-180.).

The samples were subjected to nucleic acid extraction using QIAGEN nucleic acid extraction kit, and qPCR was performed as a template to verify the specificity of the designed primers and probes according to the reaction system and the reaction conditions shown in Table 1. The qPCR amplification curve is shown in figure 1, a typical amplification curve with a Ct value of less than 35 appears in pUC57-ASFV-p72 linearized plasmid group, and the Ct value of amplification with nucleic acid of other bacteria or viruses as a template is more than 38 and has no typical amplification curve, which indicates that the primer and the probe have strong design specificity and can be used for detecting ASFV-p 72.

Optimization test of ASFV-p72 primer and probe concentration

In this example, pUC57-ASFV-p72 linearized plasmid was selected as a template, qPCR was performed using the primers shown in SEQ ID Nos. 1 to 2 and the probes shown in SEQ ID No.3 at concentrations of 0.4. mu.M, 0.8. mu.M, and 1.6. mu.M, respectively, and the concentrations of the primers and probes of ASFV-p72 were optimized according to the experimental conditions shown in Table 2.

TABLE 2 ASFV-p72 primer, optimization test grouping of Probe concentration

Experimental groups	Forward primer (μ M)	Reverse primer (mu M)	Probe (mu M)
				1	0.4	0.4	0.4
2	0.8	0.8	0.8
				3	1.6	1.6	1.6
4	0.4	0.4	0.8
				5	0.4	0.4	1.6

FIG. 2 is a graph showing the amplification curve of qPCR using primers and probes at different concentrations, and FIG. 3 is a graph showing the amplification curve of qPCR using primers at 0.4. mu.M and different probe concentrations. FIG. 2 shows that increasing primer concentration has no effect on Ct value size of qPCR reaction, therefore optimal primer concentration is selected to be 0.4. mu.M; FIG. 3 shows the amplification curves that the RFU values for 0.4. mu.M probe concentration are small compared to the RFU values for 0.8. mu.M and 1.6. mu.M probe concentrations, and between the 0.8. mu.M and 1.6. mu.M probe concentration groups, the optimal RFU value is 0.8. mu.M.

Optimization test of annealing temperature in ASFV-p72 qPCR reaction conditions

In the embodiment, pUC57-ASFV-p72 linearized plasmid is used as a template, qPCR is carried out in a reaction system with the concentration of a primer shown in SEQ ID NO.1-2 being 0.4 mu M and the concentration of a probe shown in SEQ ID NO.3 being 0.8 mu M, and eight annealing temperature gradients of 53-63 ℃ are respectively selected as the annealing temperature. The amplification curve is shown in FIG. 4, and the optimal annealing temperature was selected to be 57 ℃.

Sensitivity test for detecting ASFV-p72 by quinquagenarian PCR (quantitative polymerase chain reaction)

The linearized pUC57-ASFV-p72 plasmid is diluted by 10 times of sterile water in a gradient way and then used as a template, an optimal qPCR reaction system and reaction conditions are adopted to carry out sensitivity test, and a standard curve equation of p72 copy number and Ct is constructed. The results in fig. 5A show a typical amplification curve and have a good linear relationship (R2 ═ 0.9986, as shown in fig. 5B), indicating that the primers and probes are feasible for detecting ASFV-p72, the curve equation is-3.617 x +39.40, and the lowest detection limit is 10 copies/. mu.l.

Example 2: optimization of PMA-qPCR (polymerase chain reaction) -based method for detecting African swine fever virus infectivity

According to the invention, the PMA is adopted to pretreat the sample to be detected, and the exposed DNA is stably crosslinked through the PMA so that PCR reaction can not occur, thus the interference caused by the exposed DNA is removed, and the purpose of detecting whether the sample contains infectious ASFV is achieved. The detection principle of PMA-qPCR is shown in FIG. 6. To illustrate the feasibility of the method and to optimize the assay conditions, PMA pretreatment conditions were determined by optimizing PMA concentration and photolysis time using heat in one of the African swine fever virus inactivation modes recommended by the world animal health Organization (OIE). This example should not be considered as being able to detect only heat inactivated african swine fever virus. Based on similar principles and optimization, it is also within the scope of the present invention to determine whether African swine fever virus is infectious or not after other inactivation modes (e.g., using disinfectants). The method comprises the following specific steps:

a pig whole blood sample positive in p72 nucleic acid detection is selected, heated at 70 ℃ for 30min for inactivation treatment, centrifuged at 10,000g for 10min, and then the supernatant is collected as a test sample. Respectively transferring test samples with equal volume, adding PMA (0,10,20,40,50,60,80,100 μ M) with different concentrations, incubating at room temperature in dark place for 15min, immediately transferring the EP tube to ice after incubation is finished, and then using PMA-Lite^TMThe LED photolyzer photolyzes the sample for 15min, and the sample is continuously shaken in the period. Extracting DNA of a PMA pretreatment sample by adopting a commercial nucleic acid extraction kit,and using the copy number as a template to carry out qPCR to detect the copy number of the p72 in the sample. The difference value of the Ct of the PMA pretreatment group without PMA pretreatment and different concentrations of PMA pretreatment, namely the delta Ct, is compared, and the minimum PMA concentration with the delta Ct difference being maximum or the Ct of the PMA pretreatment group being more than 38 and without a typical amplification curve is selected as the optimal PMA working concentration. As a result, as shown in FIG. 7, the optimum working concentration of PMA was 50. mu.M.

Example 3: PMA-qPCR detection of African swine fever virus in pig positive blood sample subjected to heating inactivation treatment

Selecting a pig whole blood sample with positive p72 nucleic acid detection, transferring a proper amount of the pig whole blood sample into a plurality of parts, heating and inactivating the blood sample at 70 ℃ for 30min, centrifuging the sample for 10min by adopting 10000g, and collecting supernatant as a test sample. Meanwhile, samples which are not heated and inactivated are used as a control, PMA (50 mu M) pretreatment is carried out on the test samples under the optimized PMA-qPCR condition, the copy number of p72 is detected after nucleic acid is extracted respectively, a group without PMA pretreatment is used as a reference control, and a group with the same volume of sterilized water as a template is used as a negative control. The results are shown in Table 3.

TABLE 3 PMA-qPCR detection of Ct value of ASFVp72 in Heat-treated Positive blood samples from pigs

The data in Table 3 show that there is almost no difference in Ct values between the PMA treatment and the non-PMA treatment (both 28), while the difference between the QPCR Ct value of PMA pre-treatment and the corresponding QPCR Ct value without PMA pre-treatment (32), delta Ct of greater than 6, is found in the samples treated at 70 deg.C for 30 min. The result shows that the kit disclosed by the invention has the effect of quickly detecting the infectivity of the African swine fever virus.

Example 4: detection of p72 copy number change by combining ASFV (advanced specific immune deficiency syndrome) and qPCR (quantitative polymerase chain reaction) method for pig primary pulmonary macrophage culture and detection of African swine fever virus in heated and inactivated pig positive blood sample

In this example, the result of detection by the PMA-qPCR reagent method in example 3 was verified by a classical ASFV method, i.e., it was examined whether the infectivity of the heat-inactivated and non-heat-inactivated samples at 70 ℃ for 30min was consistent with the result of detection by PMA-qPCR.

The result of detecting ASFV infectivity by PMA-qPCR is verified by adopting a classical method of cell culture ASFV, and the specific steps are as follows: (1) separating porcine Primary Alveolar Macrophages (PAM) from alveoli of young pigs at 3-4 weeks, and culturing with DMEM (DMEM) containing 10% fetal calf serum and 1% penicillin-streptomycin antibiotic based on 5% CO₂And culturing in an incubator at 37 ℃ until the cells adhere to the wall. (2) And (3) passing the inactivated sample to be detected, a positive control (solution with infectious ASFV) and a negative control (solution without ASFV) through a sterile 0.22 mu m filter membrane, and inoculating a proper amount of the liquid into a culture solution containing PAM cells. (3) 200. mu.l of the supernatant on day 0 was collected, and the cell culture supernatant and the cells were collected after 8 days of culture, respectively.

Extracting DNA by using a commercial nucleic acid extraction kit, and detecting the content of p72 in the extracted DNA by using a qPCR method. If the Ct of the extracted nucleic acid sample is greater than 38 and has no typical amplification curve in the negative control (without ASFV solution) cells and the culture supernatant, and the Ct of the extracted nucleic acid sample is less than or equal to 35 and has a typical amplification curve in the positive control (inoculated with infectious ASFV solution) cells and the culture supernatant, the sample is determined to contain infectious African swine fever virus, and the virus has the capability of proliferating after infecting PAM cells. If the Ct value of p72 in the supernatant of the heat treatment group has no significant difference between day 0 and day 8, or p72 Ct detected in DNA extracted from cells at day 8 is more than 38 and has no obvious amplification curve, the sample to be detected is considered to be completely heat inactivated without infection ASFV; if the Ct value of the DNA extracted from the supernatant and the cells on the 8 th day is smaller than the Ct value of the DNA extracted from the cells on the 0 th day, or the Ct value in the cells is measured to be less than or equal to 35 and an obvious amplification curve exists, the sample to be detected is considered to be incompletely heated and inactivated, and infectious ASFV exists. The test results are shown in table 4.

TABLE 4 CqPCR detection of Ct value of p72 8 days after ASFV infection with PAM cells in heat-treated pig positive blood sample

The data in Table 4 show that after the sample is inoculated and inactivated by heating at 70 ℃ for 30min, the number of ASFV in cells and supernatant is not increased by PCR measurement, which indicates that the virus in the sample is inactivated. And the number of ASFV in cells and supernatant is obviously increased (Ct is reduced) by PCR determination after inoculating the unheated inactivated sample, which indicates that the virus in the sample has infectivity. The result is consistent with the detection result of PMA-qPCR, and the PMA-qPCR kit is proved to be feasible in detecting the infectious African swine fever virus.

Other non-heat inactivation means, such as chemical disinfectants, most of which contain surfactants or have strong oxidizing power, mainly act to destroy envelopes and capsids, etc., on which viral infection depends. Therefore, the integrity of the African swine fever virus capsid is destroyed, so that the nucleic acid which can permeate or directly contact the interior of the virus, such as PMA, and the like, can be crosslinked. Thus, the kit of the invention is also fully applicable in principle. The kit can complete the detection of ASFV infectivity within 2h, has the advantages of rapidness, simplicity, high efficiency and the like, and can be used for the initial screening of the rapid detection of the infectious ASFV.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

<120> PCR primer, probe, kit and detection method for detecting African swine fever virus infectivity

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

agtggaaggg tatgtaag 18

<210>2

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gcagagataa gctttcag 18

<210>3

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cagctcttcc agacgcatgt tc 22

<210>4

<211>600

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ttaggtactg taacgcagca cagctgaacc gttctgaaga agaagaaagt taatagcaga 60

tgccgatacc acaagatcag ccgtagtgat agaccccacg taatccgtgt cccaactaat 120

ataaaattct cttgctctgg atacgttaat atgaccactg ggttggtatt cctcccgtgg 180

cttcaaagca aaggtaatca tcatcgcacc cggatcatcg ggggttttaa tcgcattgcc 240

tccgtagtgg aagggtatgt aagagctgca gaactttgat ggaaatttat cgataagatt 300

gataccatga gcagttacgg aaatgttttt aataataggt aatgtgatcg gatacgtaac 360

ggggctaata tcagatatag atgaacatgc gtctggaaga gctgtatctc tatcctgaaa 420

gcttatctct gcgtggtgag tgggctgcat aatggcgtta acaacatgtc cgaacttgtg 480

ccaatctcgg tgttgatgag gattttgatc ggagatgttc caggtaggtt ttaatcctat 540

aaacatatat tcaatgggcc atttaagagc agacattagt ttttcatcgt ggtggttatt 600

Claims

1. The PCR primer and the probe for rapidly detecting the infectivity of the African swine fever virus are characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.

2. The use of the PCR primers and probes according to claim 1 for detecting African swine fever virus and for preparing a standardized kit for detecting African swine fever virus.

3. A kit for rapidly detecting the infectivity of African swine fever virus is characterized by comprising: 1) the PCR primers and probes as claimed in claim 1, 2) a nucleic acid dye that binds to nucleic acids and has light sensitive properties.

4. The kit for rapidly detecting the infectivity of African swine fever virus according to claim 3, wherein the nucleic acid dye which is combined with nucleic acid and has photosensitive property is azido propidium bromide.

5. The kit for rapidly detecting the infectivity of African swine fever virus according to claim 3, wherein the kit further comprises Taq DNApolymerase, Buffer, dNTP and sterilized water.

6. The method for rapidly detecting the infectivity of African swine fever virus based on the kit of any one of claims 3-5, which comprises the following steps:

(1) taking a sample to be detected, dividing the sample into 2 parts in equal volume, wherein 1 part is subjected to photosensitive nucleic acid dye pretreatment, and the other part is added with sterilized water or buffer solution to serve as a comparison sample;

(2) respectively extracting total DNA of two samples to be detected;

(3) respectively taking the two DNAs extracted in the step (2) as templates to carry out qPCR real-time fluorescent quantitative detection; the nucleotide sequence of the primer in the qPCR reaction is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQID NO. 3;

(4) and after the qPCR reaction is finished, rapidly detecting whether the infectious African swine fever virus exists in the sample according to the amplification curve and the cycle threshold of the two nucleic acid samples in the fluorescent quantitative PCR reaction.

7. The method for rapidly detecting the infectivity of African swine fever virus according to claim 6, wherein the steps of pre-treating the sample to be detected with the photosensitive nucleic acid dye comprise the following steps:

8. The method for rapidly detecting the infectivity of African swine fever virus according to claim 6, wherein the qPCR reaction system is: 0.5 mul of Taq DNApolymerase, 2 mul of 10 XBuffer, 1.6 mul of 2.5mM dNTP, 0.4 mul of 20 mul of a primer shown by SEQ ID NO.1, 0.4 mul of a primer shown by 20 mul SEQ ID NO.2, 0.8 mul of a probe shown by 20 mul SEQ ID NO.3, 2 mul of a template and 12.3 mul of sterilized water; the 10 × buffer comprises: 200mM Tris-HCl (pH 8.3), 200mM KCl, 100mM (NH)₄)₂SO₄，20mM MgSO₄And 5 wt% NP 40.

9. The method for rapidly detecting the infectivity of African swine fever virus according to claim 6, wherein the qPCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, and then circulation for 40 times; the procedure for each cycle was: denaturation at 95 ℃ for 5sec, annealing at 57 ℃ for 30sec, and collecting the FAM fluorescence channel signal, and after the cycle is completed, the reaction is terminated.

10. The method for rapidly detecting the infectivity of African swine fever virus according to claim 6, wherein the standard for detecting the presence or absence of the infectious African swine fever virus in the sample is: (1) the Ct value of a sample treated by the photosensitive nucleic acid dye is larger than that of a sample not pretreated by the dye, and the sample contains inactivated ASFV; (2) the larger the Δ Ct, the higher the inactivated virus content; (3) and if the delta Ct is more than or equal to 6 or the qPCR reaction of the sample group treated by the photosensitive nucleic acid dye has no typical amplification curve and the Ct is more than 38, the virus in the sample is completely inactivated.