CN113462817A - PCR primer, probe, kit and detection method for detecting African swine fever virus capsid integrity - Google Patents
PCR primer, probe, kit and detection method for detecting African swine fever virus capsid integrity Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to African swine fever virus detection with a complete capsid structure. Aiming at the problems that the lowest detection limit of a detection method capable of identifying infectious ASFV is 10 copies/mu l and the sensitivity is not high enough in the existing detection method of ASFV, the invention provides a PCR primer and a probe for detecting the integrity of African swine fever virus capsid, 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. The detection kit disclosed by the invention is accurate and reliable in detection result and simple and convenient to operate, can be used for large-scale farms, laboratories and epidemic disease prevention and control units, and has important significance for prevention and control of African swine fever in actual production.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to African swine fever virus detection with a complete capsid structure.
Background
African Swine Fever (ASF) is a swine acute febrile highly-contagious disease caused by African Swine Fever Virus (ASFV), and clinical symptoms are similar to swine fever, mainly manifested by fever, skin cyanosis, bleeding of lymph nodes, kidneys, gastrointestinal tract, etc. Virulent strains can cause high mortality of pigs, and the death rate reaches more than 90%. At present, effective vaccines and medicines are not available for the disease, the most effective prevention and control measure is quarantine, and the prevention and control of the African swine fever at the level of a farm is a system engineering based on biological safety. Therefore, it is imminent to improve the biosafety level of the farm in China, remove the existing ASFV and effectively prevent the ASFV from reentering the farm.
Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qPCR) is a new Quantitative test technology introduced by Applied Biosystems in the United states in 1996, and is characterized in that a PCR product is marked and tracked through a fluorescent dye or a fluorescent marked specific probe, the reaction process is monitored on line in Real time, the product can be analyzed by combining with corresponding software, and the initial concentration of a sample template to be detected is calculated. In 2019, 1 month and 4 days, the first batch of ASFV on-site rapid detection reagents are published by the rural part of China agriculture, and more than 60 percent of the ASFV on-site rapid detection reagents adopt a real-time qPCR detection method. The method can detect the reaction process in real time, determine the absolute quantity of the template, has strong specificity and the like, so that the qPCR has higher utilization value in the actual ASFV investigation. However, the qPCR detection result being greater than the threshold value only indicates that the target nucleic acid fragment exists in the sample, and cannot reflect that the target fragment is derived from free nucleic acid or complete virus, and cannot judge whether the sample contains infectious virus. There is no guidance for determining whether a sample of a swine with clinical symptoms in the onset phase contains infectious virus, but for environmental samples, particularly in the case of re-feeding, determining whether a positive sample has a complete capsid virus is one of the prerequisites for assessing whether the field is re-enabled.
The existing detection methods for the African swine fever virus are various, such as a common PCR method, a fluorescent RAA method, a high-sensitivity fluorescence immunoassay method, a sandwich Elisa antigen detection method, an indirect Elisa antibody detection method, a blocking Elisa antibody detection method, a sandwich Elisa antibody detection method, an indirect immunofluorescence method and the like, but the detection methods can only detect whether virus nucleic acid exists, and cannot judge whether a virus capsid is complete or not and whether the virus capsid has infectivity or not. If the virus is to be determined to be infectious, cell experiments or animal experiments are required, but the experiments are complicated to operate and are easily affected by individual differences of recipient cells or recipient animals. Whether virus has infectivity needs to be detected in a plurality of aspects such as an experiment for detecting the killing rate of a disinfectant, an environment detection experiment for recultivation in an old farm, an experiment for detecting whether the pork with the virus flowing in the market has infectivity, and the like, and the existing technology can not meet the experimental requirements. The prior detection technology can detect infectious viruses, the sensitivity of a probe and a primer sequence can reach the lowest detection limit of 10 copies/mu l, the detection sensitivity can not reach a certain lower limit, and the detection requirements of certain detection items can not be met.
The PMA-PCR technology, also called vitality PCR (vPCR), can be used for detecting whether bacteria or viruses have complete structures or not, can be used for researching the physicochemical characteristics of the viruses, screening the types and the optimal action conditions of disinfectants and evaluating the effect of biological safety measures, simultaneously eliminates the interference of free nucleic acid on results in the detection process, and has great application potential in the prevention and control of ASFV. The technical principle is established on the action mechanism of PMA, Propidium bromide azide (PMA) is an azide type nucleic acid dye, has high affinity to nucleic acid, can enter the interior of an inactive virus with damaged virus capsid (lipoprotein), and under the action of strong light, a carried photosensitive azide group is converted into a high-activity nitrene free radical and reacts with any hydrocarbon near a binding site to form a stable covalent nitrogen-carbon bond, so that the permanent modification of a nucleic acid molecule is realized, and the amplification of the nucleic acid molecule is blocked, and the influence of the inactive virus nucleic acid on a detection result is eliminated; meanwhile, since the active virus has a complete capsid, PMA is blocked outside the virus, and thus amplification of nucleic acid molecules of the active virus cannot be blocked. In addition, PMA residing in solution without cross-linking with nucleic acid molecules reacts with water molecules to form inactive hydroxylamine upon irradiation with intense light.
Patent CN 110894556 a discloses a method for detecting whether there is infectious ASFV particles in a sample by using azide propidium bromide (PMA) in combination with qPCR, which utilizes PMA which cannot penetrate the envelope structure of a virus but can non-specifically modify nucleic acid molecules so that they cannot be amplified by PCR or qPCR, and can be used for identifying ASFV with complete envelope structure. However, the accuracy and sensitivity of the method are still further improved. In addition, the method is used for identifying whether the ASFV particles have infectivity, wherein the infectivity is determined by pathogenicity of pathogens, autoimmune state of a host, breeding environment, breeding management and the like, and even if the pathogens with complete capsids do not have the infectivity necessarily but cause other risks. Therefore, the method can screen a part of the capsid with complete pathogens, and the accuracy is still to be improved.
The African swine fever virus enters a farm through water sources, feeds or feeders, vehicles, staff foods and the like, the virus content is very low, the virus is generally difficult to detect, the lowest limit of the existing method for detecting the infectious virus is 10 copies/mu l, the detection sensitivity is still to be improved, and the development of an ASFV detection method which has high detection sensitivity and can identify whether the virus has infectivity is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing detection method for detecting ASFV has the problems that the lowest detection limit of the detection method for identifying infectious ASFV is 10 copies/mu l, and the sensitivity is not high enough.
The technical scheme for solving the technical problems comprises the following steps: provides a PCR primer and a probe for detecting the integrity of the African swine fever virus capsid.
The invention solves the first technical problem by providing a PCR primer and a probe for detecting the integrity of the African swine fever virus capsid, 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.
Upstream primer for detecting African swine fever virus capsid integrity by SEQ ID NO.1
CGCTCATGATGCTACCAATATC。
Downstream primer for detecting African swine fever virus capsid integrity by SEQ ID NO.2
GGATGGATAGTCCTGTAAGATTTC。
Probe for detecting African swine fever virus capsid integrity by SEQ ID NO.3
5’HEX-ACATCGTGAACAAGTATATCGGCAACCTG-3’。
Furthermore, the 3' of the probe also contains a fluorescence quencher BHQ 1.
The invention solves the second technical problem by providing the application of the PCR primer and the probe for detecting the integrity of the African swine fever virus capsid in the preparation of the kit for detecting the African swine fever virus.
The third technical problem solved by the invention is to provide a kit for detecting the integrity of the African swine fever virus capsid.
Further, the kit comprises: a. primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and probes with nucleotide sequences shown as SEQ ID NO. 3; b. a nucleic acid dye that binds to nucleic acids and has a light sensitive property.
Further, in the kit for detecting the integrity of the African swine fever virus capsid, the nucleic acid dye is propidium azide bromide.
Further, the kit for detecting the integrity of the African swine fever virus capsid specifically comprises: PMA, 2 XPro TaqHS Probe Premix, Probe, upstream primer, downstream primer, template and sterile water.
Furthermore, the kit for detecting the integrity of the African swine fever virus capsid specifically comprises the following components: PMA 200. mu.L, 2 XPRO TaqHS Probe Premix 12.5. mu.L, 10 pmol/. mu.L Probe 1. mu.L, 10 pmol/. mu.L upstream primer 1.2. mu.L, 10 pmol/. mu.L downstream primer 1.2. mu.L, template 2. mu.L, sterile water 7.1. mu.L.
The fourth technical problem to be solved by the invention is to provide a method for detecting the integrity of the African swine fever virus capsid by using the kit, which comprises the following steps:
a. taking a sample to be detected, dividing the sample into 2 parts, carrying out PMA treatment on one part, and carrying out sterile water treatment on the other part;
b. extracting DNA of 2 samples respectively;
c. carrying out qPCR detection on the DNA extracted in the step b;
d. and after the qPCR reaction is finished, rapidly detecting the integrity of the capsid of the African swine fever virus in the sample according to the amplification curve and the cycle threshold of the fluorescent quantitative PCR reaction of the two nucleic acid samples.
Wherein, in the method for detecting African swine fever virus, the sample to be detected in the step a is blood or tissue.
Furthermore, the tissue needs to be pre-processed, and the operation is as follows: processing tissue with liquid nitrogen, grinding into fine powder, adding normal saline, grinding uniformly, centrifuging at 8000r/min for 3min, and collecting supernatant.
Wherein, in the method for detecting African swine fever virus, the specific operation of PMA treatment in the step a is as follows: adding PMA into the sample, shaking by a vortex oscillator for 30s, mixing uniformly, standing for 20min in dark, and performing light activation reaction for 20 min.
In the method for detecting African swine fever virus, the light activation refers to irradiating the sample with halogen lamp.
Wherein, in the method for detecting African swine fever virus, the reaction system for qPCR detection comprises: 2 XPro TaqHS Probe Premix 12.5. mu.L, 10 pmol/. mu.L Probe 1. mu.L, 10 pmol/. mu.L upstream primer 1.2. mu.L, 10 pmol/. mu.L downstream primer 1.2. mu.L, template 2. mu.L, sterile water 7.1. mu.L.
Wherein, in the method for detecting African swine fever virus, the reaction conditions of qPCR detection are as follows: and (3) at 95 ℃ for 3min, then at 95 ℃ for 15s and at 55 ℃ for 30s, performing 40 cycles in the last two steps, and performing fluorescence signal detection in the annealing stage.
The detection method comprises the following steps:
(1) the Ct value of the HEX channel in the detection result of the detected sample is less than or equal to 38, the amplification curves are typical S curves, the result is reported to be positive to the African swine fever virus, and the virus capsid has a complete structure and is infectious;
(2) the value of the Ct of the HEX channel 38 in the detection result of the detected sample is less than or equal to 40, the sample is judged to be suspicious, the suspicious sample is required to be detected again, if the value of the Ct is still 38 less than or equal to 40 after the detection is repeated, the amplification curves are typical S curves, the result is reported to be positive to the African swine fever virus, and the virus capsid structure is complete and has infectivity;
(3) the HEX channel in the detection result of the detected sample has no Ct value or no typical S-type amplification curve, and the African swine fever virus infectivity is reported to be negative, namely the African swine fever virus has no infection capacity.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a primer, a probe, a kit and a method for detecting the integrity of an African swine fever virus capsid, the lowest detection virus amount can reach 5.18 copies/mu l, compared with the existing lowest 10 copies/mu l, the detection limit is further reduced, the detection sensitivity is higher, and the detection requirements of more detection items can be met. The detection method is suitable for the fields of screening the African swine fever virus disinfectant, detecting whether the flowing meat food has infectivity, detecting whether the internal and external environments, water sources, feeds, vehicles entering and exiting, personnel and materials of a pig farm have live toxicity, evaluating and optimizing biological safety measures, evaluating whether an old disease farm reaches a re-breeding safety level and the like.
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FIG. 1 shows the PCR identification result of recombinant plasmid standard, M: DL2000 DNAmarker; 1: an MGF505 gene fragment; 2: negative control;
FIG. 2 shows a standard curve of a fluorescent quantitative PCR reaction;
FIG. 3 shows the results of the sensitivity test of the method of the present invention;
FIG. 4 shows the results of the specificity test of the method of the present invention.
Detailed Description
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
Used in the examples: (1) a plasmid containing ASFV MGF505_1R truncated gene sequence; standard plasmids of porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, parvovirus, Japanese encephalitis B, porcine epidemic diarrhea virus, classical swine fever virus and foot and mouth disease virus are provided by key laboratories of Sichuan province of animal epidemic diseases and human health.
(2) DH5 alpha competent cells, viral genome DNA extraction kit, common agarose gel DNA recovery kit, common plasmid mini-extraction kit were purchased from Tiangen Biotechnology Ltd.
(3) pMD19-T vector, 2 XTAQQ PCR Master Mix, DL2000 DNAmarker, etc. were purchased from Bao bioengineering (Dalian) Co., Ltd., and Azide propidium bromide (PMA) was purchased from biotin.
(4) The rest of the reagents are common commercial products.
Example 1PCR primer and Probe design and Synthesis
Primers and fluorescent probes specific to MGF505_1R gene (Table 1) were designed using AllelelD 6.0 design software with reference to the nucleic acid sequence of ASFV Pig/CN/HLJ/2018(GenBank: MK333180.1), and the primers and probes were synthesized by Shanghai Biotechnology, Inc. The length of the target fragment gene is 103bp, and the target fragment gene is positioned at 29213-29234 nucleotides of ASFV Pig/CN/HLJ/2018.
TABLE 1 primer and TaqMan Probe sequence information
Example 2 construction and identification of recombinant plasmid standards
A103 bp gene sequence of a target fragment (nucleotide sequence 29213-29234 of accession number MK333180.1) was artificially synthesized based on the MGF505 gene sequence of ASFV on GenBank as a positive template for the subsequent experiments. And performing amplification culture on the template. The specific primer MGF505-F/MGF505-R is adopted to amplify the target gene, the target fragment is recovered after 1 percent agarose gel electrophoresis detection, the target fragment is cloned to a pMD19-T vector, and the positive clone product is sequenced and identified by Shanghai biological engineering technical service company Limited. Culturing positive bacterial strain, extracting recombinant plasmid with plasmid extracting kit and named pMD19-ASFV-MGF 505. The concentration of the recombinant plasmid standard was determined using a NanoDrop 2000 nucleic acid protein analyzer and the copy number was calculated. According to the formula: recombinant plasmid copy number (copies/. mu.L) ═ plasmid concentration × 10-9×6.02×1023) V (660 daltons/base x number of bases), the copy number is calculated.
The MGF505 gene of the synthetic strain was amplified by PCR using specific primers. As a result, the strains were amplified to obtain bands of 103bp each (as shown in FIG. 1). And recovering and purifying the PCR products, and cloning the PCR products to pMD19-T vectors respectively to construct a recombinant plasmid standard pMD19-ASFV-MGF 505. As a result of sequencing, no MGF505 gene mutation was found compared with the reference sequence (accession number: MK333180.1), and the result was in line with the expectation. The plasmid standard pMD19-ASFV-MGF505 is correctly constructed, and the plasmid standard is calculated to be 1.3 multiplied by 10 respectively10copies/μL。
Example 3 construction of fluorescent quantitative PCR reaction System
A25 mu L reaction system is adopted, 2 mu L positive plasmids with the same concentration are used as templates, primers and probes with different concentrations are selected, the optimal concentrations of the primers and the probes are optimized by a matrix method, the lowest Ct value and the highest fluorescence intensity increase value (delta Rn) of the reaction are obtained, the reaction amplification efficiency and sensitivity are improved, and the annealing temperature (50-60 ℃) is optimized. The optimized fluorescent quantitative PCR reaction system has a total volume of 25 μ L, which comprises 2 XPro TaqHS Probe Premix 12.5 μ L, upstream and downstream primers (10 pmol/. mu.L) each 1.2 μ L, Probe (10 pmol/. mu.L) 1 μ L, template DNA2 μ L, and sterile water is added to 25 μ L. The Bio-Rad CFX connect fluorescent quantitative PCR instrument amplification program is as follows: pre-denaturation at 95 ℃ for 3 min; 15s at 95 ℃; 30s at 55 ℃; the last two steps are carried out for 40 cycles, the fluorescence signal detection is carried out in the annealing stage, and the fluorescence channel is selected to be HEX.
The constructed pMD19-T recombinant plasmid is used as a standard substance for pMD19-ASFV-MGF505 quantification, the standard substance is diluted by 10 times of gradient according to the determined plasmid concentration, and 8 gradients (10) are carried out in total3～1010) Diluting. And (3) performing fluorescent quantitative PCR by taking the recombinant plasmids with different concentrations as templates, recording the Ct value of each gradient standard substance, and establishing a quantitative standard curve of the corresponding relation between the copy number of the plasmid and the copy number of the plasmid. The optimized fluorescent quantitative PCR reaction system is used for 8 dilutions (1.3X 10)3～1.3×1010copies/mu L) with a standard curve of 1.3 multiplied by 103-1.3×1010The copy number has a good linear relation, the correlation coefficient is 0.998, and a linear equation of the copy number and the Ct value of the standard product is obtained (shown in figure 2).
Example 4 sensitive, specific, reproducible detection of the methods of the invention
And (3) sensitivity test:
by ddH2O10-fold serial dilution of the standard to 1.3X 100.6-1.3×1078 gradients of copies/mu L are respectively used as templates, the detection is carried out under the optimal reaction condition, the minimum detection quantity of the established fluorescence quantitative PCR detection method is determined, and the method is used for evaluationThe sensitivity of (2).
The diluted 8 gradients (1.3X 10)0.6-1.3×107copies/mu L) standard substance as template, detecting with optimal reaction condition, and determining the minimum detection amount of the established fluorescent quantitative PCR detection method to be 1.3 multiplied by 100.6copies/. mu.L (5.18 copies/. mu.L), as shown in FIG. 3, it was found that the method had high sensitivity.
And (3) specificity test:
extracting standard plasmid DNA of porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, parvovirus, Japanese encephalitis B, porcine epidemic diarrhea virus, hog cholera virus and foot and mouth disease virus as a template, simultaneously taking a recombinant plasmid standard pMD19-ASFV-MGF505 as a positive control, and ddH as a negative control2O is used as a negative control, and the fluorescence quantitative PCR method established in the research is adopted for amplification to evaluate the specificity of the method (as shown in figure 4).
And (3) repeatability test:
at 1.3X 104、1.3×105、1.3×106Taking 3 concentrations of recombinant plasmids of copies/mu L as detection templates, carrying out fluorescent quantitative PCR amplification on the recombinant plasmids under the same reaction condition, repeatedly detecting each concentration of template for 3 times, and calculating the average and standard deviation by adopting SPSS statistical software to evaluate the repeatability of the method.
This study is on a 1.3X 104～1.3×106The 3 concentrations of recombinant plasmids, copies/. mu.L, were tested, 3 replicates per concentration were performed, and the Ct values obtained were counted and the mean, standard deviation calculated. The results are shown in Table 2. As can be seen from Table 2, the fluorescence quantitative PCR detection method for the African swine fever MGF505 genotype established by the test has better repeatability, and can stably and reliably detect the African swine fever MGF505 genotype sample.
TABLE 2 fluorescent quantitative PCR repeatability test results
From the examples it can be seen that: the invention develops the PCR primer, the probe, the kit and the method for detecting the integrity of the African swine fever virus capsid, has strong sensitivity, good specificity and repeatability, can detect the African swine fever virus capsid with the lowest limit of 5.18 copies/mu l, further improves the detection sensitivity, and is suitable for popularization and use.
Claims (10)
1. PCR primers and probes for detecting the integrity of the African swine fever virus capsid are characterized in that: the nucleotide sequences of the primers are 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 PCR primers and probe for detecting the integrity of the capsid of african swine fever virus according to claim 1, wherein: the 3' of the probe also contains a fluorescence quencher BHQ 1.
3. Use of the PCR primers and probes for detecting the integrity of the african swine fever virus capsid according to claim 1 or 2 for the preparation of a kit for detecting the african swine fever virus.
4. Kit comprising the PCR primers and probes for detecting the integrity of the African swine fever virus capsid according to claim 1 or 2.
5. The kit of claim 4, wherein: the kit comprises: a. primers with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and probes with nucleotide sequences shown as SEQ ID NO. 3; b. a nucleic acid dye that binds to nucleic acids and has a light sensitive property.
6. The kit of claim 4, wherein: the nucleic acid dye is azido propidium bromide.
7. The kit according to any one of claims 4 to 6, characterized in that: the kit comprises the following specific components: PMA, 2 XPro TaqHS Probe Premix, Probe, upstream primer, downstream primer, template and sterile water.
8. The kit of claim 7, wherein: the kit comprises the following specific components: PMA 200. mu.L, 2 XPRO TaqHS Probe Premix 12.5. mu.L, 10 pmol/. mu.L Probe 1. mu.L, 10 pmol/. mu.L upstream primer 1.2. mu.L, 10 pmol/. mu.L downstream primer 1.2. mu.L, template 2. mu.L, sterile water 7.1. mu.L.
9. The method of using the kit of any one of claims 4 to 8, comprising the steps of:
a. taking a sample to be detected, dividing the sample into 2 parts, carrying out PMA treatment on one part, and carrying out sterile water treatment on the other part;
b. extracting DNA of 2 samples respectively;
c. carrying out qPCR detection on the DNA extracted in the step b;
d. and after the qPCR reaction is finished, rapidly detecting the integrity of the capsid of the African swine fever virus in the sample according to the amplification curve and the cycle threshold of the fluorescent quantitative PCR reaction of the two nucleic acid samples.
10. The method of using the kit according to claim 9, wherein: the reaction conditions of the qPCR detection are as follows: and (3) at 95 ℃ for 3min, then at 95 ℃ for 15s and at 55 ℃ for 30s, performing 40 cycles in the last two steps, and performing fluorescence signal detection in the annealing stage.
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