CN114540549A - Primer, probe, kit and iPCR method for detecting African swine fever virus - Google Patents
Primer, probe, kit and iPCR method for detecting African swine fever virus Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology detection, and discloses a primer, a probe, a kit and an iPCR method for detecting African swine fever virus; wherein the primers are divided into an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the kit comprises the primer and the probe. The kit has the advantages of short detection time, reaction time of only 42 minutes, strong specificity, high sensitivity and convenient storage, can meet the requirement of rapid detection of the African swine fever virus in field and field environments by being matched with a constant-temperature isolated PCR instrument, has important clinical use value, and can be widely popularized at the basic level.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a primer, a probe, a kit and an iPCR method for detecting African swine fever viruses.
Background
The African Swine Fever Virus (ASFV) is the only member of the African swine fever related virus family, and is clinically characterized by acute, febrile, hemorrhagic, high morbidity and high mortality, and the epidemic situation of the African swine fever caused by the virus has spread to all countries around the world, which causes serious attack to the pig industry of the epidemic situation generating countries. Because the clinical symptoms of ASF are difficult to distinguish from Classical Swine Fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS), and there is no mature and effective ASF vaccine or cure regimen at present, rapid diagnosis and timely eradication become the main measures for controlling the epidemic.
Clinical detection of African swine fever virus mainly depends on common PCR, real-time fluorescence PCR and other laboratory diagnosis methods, but the existing detection method has the technical defects of long time consumption, low sensitivity, complex detection steps, high equipment requirement and the like.
Disclosure of Invention
In order to solve the technical defects, the invention provides a primer, a probe, a kit and an iPCR method for detecting the African swine fever virus, and the method has the characteristics of short detection time, strong specificity, high sensitivity, convenience in storage, simplicity, rapidness and practicability, and can meet the rapid detection requirement of the African swine fever virus in field and field environments.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer and a probe for detecting African swine fever virus, which comprise the primer and the probe for detecting the African swine fever virus;
the primer is divided into an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
Furthermore, the 5 'end of the nucleotide sequence of the probe is connected with a fluorescent group, and the 3' end of the nucleotide sequence of the probe is connected with a non-fluorescent quenching group and a quenching group;
the fluorescent group is selected from one of FAM, TET, HEX, CY3 and JOE, and is further FAM;
the quenching group is selected from one of MGB, BHQ1, BHQ2, TAMRA, DABCYL and Eclipse, and is further MGB.
The second aspect of the invention provides a kit for detecting African swine fever virus, which comprises the primer and the probe.
Further, the kit also comprises reaction buffer solution, Taq enzyme, reverse transcriptase, a positive reference substance, a negative reference substance and dNTPs;
dNTPs consist of four nucleotides, dATP, dGTP, dCTP and dUTP.
Further, the reaction buffer consisted of the following components: 500mM KCl, 100mM Tris-HCl, 15mM MgCl2Add ddH2The total volume of O is 3ml, and the pH value is 8.0-8.5.
Further, the final concentration of Taq enzyme was 5U. mu.L-1The final concentration of reverse transcriptase was 20 U.mu.L-1。
Further, the positive control is a sample containing African swine fever virus DNA fragments;
the positive control is prepared by the following method: 100 mu L of DNA sample containing African swine fever virus DNA fragment is filled into a PCR tube, cooled to-40 to-80 ℃, and then freeze-dried under the air pressure of 8-12 pa to form dry powder.
Further, the negative control is a sample of DNA fragment of African swine fever virus;
the negative control is prepared by the following method: 100 mu L of a sample of the DNA fragment of the African swine fever virus is put into a PCR tube, cooled to-40 to-80 ℃, and then lyophilized under the air pressure of 8-12 pa to form dry powder.
Furthermore, the final concentration of the upstream primer and the final concentration of the downstream primer of the African swine fever virus are both 10 mu mol mu L-1The final concentration of the probe was 10. mu. mol. mu.L-1；
The final concentration of dNTPs was 2.5 mM.
The second aspect of the invention provides an iPCR method for detecting African swine fever virus, which comprises the following steps:
1) preparing a DNA template: extracting total DNA of the African swine fever virus, and preparing a DNA template;
2) establishing an iPCR reaction system:
adding 100 mu L of reaction buffer solution into the positive reference substance, mixing to prepare a positive reference substance, adding 50 mu L of reaction buffer solution and 5 mu L of positive reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube, and preparing to obtain a positive reference reaction system;
adding 100 mu L of reaction buffer solution into a negative reference substance freeze-drying tube to prepare a negative reference substance, and then adding 50 mu L of reaction buffer solution and 5 mu L of negative reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a negative reference reaction system;
adding 50 mu L of reaction buffer solution and 5 mu L of detection sample DNA into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a detection sample DNA reaction system;
then respectively taking 50 mu L of mixture from each reaction system and transferring the mixture into a constant-temperature isolated fluorescent PCR reaction tube;
the prepared fluorescent quantitative PCR reaction tube is instantaneously centrifuged for 5s at high speed, so that bubbles are avoided;
3) and (3) amplification detection: the PCR reaction tube is placed in a constant temperature isolated fluorescent PCR instrument for reaction, and the result shows that "+" represents positive and "-" represents negative.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection kit has the advantages of strong specificity, good stability, high sensitivity, short detection time, no pollution and no need of electrophoresis, can obtain a detection result after being detected on a computer for 42min, is suitable for field or even field rapid detection, and has good application prospect.
(2) The invention can quickly and accurately detect the African swine fever virus DNA in the detected sample, and can also detect the bovine rotavirus DNA for the molecular epidemiological investigation of the bovine rotavirus.
(3) The fluorescent quantitative PCR reaction solution comprises reverse transcriptase and Taq enzyme, so that the reverse transcription and amplification of nucleic acid are carried out in the same tube, the operation is simplified, the reagent is saved, the tube closing operation is carried out at the same time, the degradation and pollution possibility of DNA is reduced, and the cost of sample diagnosis is reduced.
(4) The fluorescent quantitative PCR reaction solution, the positive reference substance and the negative reference substance are all prepared in a freeze-drying mode, so that the storage condition of the kit is reduced, the kit can be stored for one year at 4 ℃, the transportation and the storage are convenient, and the stability of the reagent is improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the results of the sensitivity test of the positive sample of the present invention, wherein 1: 2.55X 101copies/μL；2： 2.55×100copies/μL；3：2.55×10-1copies/μL；4：2.55×10-2copies/μL。
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Example 1 African swine fever virus detection kit
The kit comprises the following components:
(1)3ml reaction buffer: prepared from 500mM KCl, 100mM Tris-HCl (pH 8.3), 15mM MgCl2And the remainder ddH2And (C) O.
(2) Fluorescent quantitative PCR reaction solution freeze-drying tube (50 tubes):
the fluorescent quantitative PCR reaction solution comprises 1 mu L of Taq enzyme per tube (5U. mu.L)-1) 1.25. mu.L/tube of reverse transcriptase (20U. mu.L)-1) 3.5. mu.L/tube (10. mu. mol. mu.L) of each of the upstream and downstream primers for detection-1) And probe 0.25. mu.L/tube (10. mu. mol. mu.L)-1) dNTPs 4. mu.L/tube (2.5 mM); and adding the mixture into a 0.5ml PCR tube, reducing the temperature to-50 ℃, and then carrying out freeze-drying for 24 hours under the air pressure of 10pa to form a dry powder, and closing the tube to prepare a fluorescent quantitative PCR reaction liquid freeze-drying tube. Wherein, the upstream and downstream primers and probes for detecting the African swine fever virus have 3 strips, and the length of the amplified African swine fever virus P72 gene is 63 bp.
The upstream primer sequence is 5'-CCTCGGCGAGCGCTTTATCAC-3', SEQ ID NO 1;
the sequence of the downstream primer is 5'-GGAAACTCATTCACCAAATCCTT-3', SEQ ID NO 2;
the probe sequence is as follows: FAM-CGATGCAAGCTTTAT-MGB, SEQ ID NO 3.
(3) Positive control freeze-dried tube (1 tube): filling 100 mu L of DNA sample containing African swine fever virus DNA fragment into a PCR tube, reducing the temperature to-50 ℃, and then freeze-drying for 24h under the air pressure of 10pa to form dry powder, and then closing the tube to prepare the African swine fever virus DNA chip; wherein, the sequence of the African swine fever virus DNA is shown as SEQ ID NO. 4.
(4) Negative control freeze-dried tube (1 tube): 100 mu L of a sample of DNA fragments of African swine fever virus is loaded into a PCR tube, the temperature is reduced to-50 ℃, and then the sample is lyophilized for 24 hours under the air pressure of 10pa to form dry powder, and the dry powder is prepared by closing the tube.
The kit is stored at 4 ℃, and ice bags are required to be added during transportation.
Example 2 method of Using African Swine fever Virus detection kit
The method comprises the following steps:
1) DNA template: taking 200 mu L of tissue sample supernatant, extracting total DNA of the detected sample supernatant by referring to the extraction specification of a PetNAD kit of Jinhong Jie (Xiamen) Biotechnology Limited company, and preparing a DNA template;
2) preparation of a fluorescent PCR reaction system: preparing a fluorescent PCR reaction system on ice;
adding 100 mu L of reaction buffer solution into a positive reference substance freeze-drying tube, mixing to prepare a positive reference substance, adding 50 mu L of reaction buffer solution and 5 mu L of positive reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube, and preparing to obtain a positive reference reaction system;
adding 100 mu L of reaction buffer solution into a negative reference substance freeze-drying tube to prepare a negative reference substance, and then adding 50 mu L of reaction buffer solution and 5 mu L of negative reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a negative reference reaction system;
adding 50 mu L of reaction buffer solution and 5 mu L of detection sample DNA into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a detection sample DNA reaction system;
then, 50. mu.L of each mixture was transferred from each reaction system to a fluorescent PCR reaction tube isolated at a constant temperature. And (4) during sample application, the operation of avoiding light is taken care of. And (3) instantly centrifuging the prepared fluorescent quantitative PCR reaction tube at a high speed for 5s to avoid bubbles.
3) And (3) amplification detection: placing the PCR reaction tube in a constant-temperature isolated fluorescent PCR instrument (POCKIT Micro), and pressing an operation key to start reaction;
4) and (3) judging a detection result: "+" indicates positive, and "-" indicates negative.
EXAMPLE 3 Standard preparation and determination of sensitivity
The total DNA of the supernatant of the sample to be examined was extracted by taking 200. mu.L of the supernatant of the tissue sample and referring to the extraction instructions of the PetNAD kit of Jinhong Jie (Xiamen) Biotech Co.
And respectively carrying out PCR amplification on the prepared DNA by using primers of African swine fever virus, and taking the corresponding pathogenic DNA as a positive control and taking no template as a negative control.
The reaction system is as follows: DNA 2.0. mu.L, 2 XTAQ PCR Master Mix 12.5. mu.L, upstream and downstream primers 1.0. mu.L each, ddH2O 8.5.5. mu.L, total volume 25. mu.L.
The PCR amplification conditions are 95 ℃ for 5min, 95 ℃ for 30s, 52 ℃ for 30s, 72 ℃ for 30s, 30 cycles and 72 ℃ for 10 min.
And identifying the PCR product through 3% agarose gel electrophoresis, recovering a target fragment by using a gel recovery kit, cloning the target fragment to a pMD19-T vector (Takara Bio-engineering Co., Ltd.), transforming escherichia coli DH5 alpha competent cells, screening positive clones, inoculating the positive clones to an LB liquid culture medium containing ampicillin, culturing for 8h at 37 ℃, extracting recombinant plasmids by using a plasmid extraction kit, and sequencing by using a Biol Co., Ltd.
And (3) taking the positive plasmid with correct sequencing as a positive standard substance of the African swine fever virus, measuring the concentration of the positive plasmid by using a nucleic acid protein detector, and calculating the nucleic acid concentration according to the following formula: concentration of positive standard (copies/. mu.L) ═ 6.02X 1023copies/mol)×(29.75ng/μl)×10-3/(1.85*106g/mol)＝2.55×1010copies/. mu.L. Positive standard was diluted 10-fold in gradient (10)-1～10-10) To 103、102、101And 100Detecting four dilution samples, putting each reaction tube into a POCKIT Micro reaction hole, pressing a running key, finishing the reaction after 42 minutes, wherein the positive result is "+" and the negative result is "-", and the results are shown in figure 1, wherein 10 are shown in figure 13、102、 101The result is "+" 100The result is "-", which indicates that the lowest detection limit of the kit is 2.55 copies/. mu.L.
Example 4 specificity and stability test
The kit developed by the invention is used for detecting 28 African swine fever virus positive samples, 10 negative samples, 6 related pathogens of porcine parvovirus, swine fever, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine epidemic encephalitis B virus and porcine epidemic diarrhea virus, and an African swine fever virus positive standard substance is set as a control to evaluate the specificity of the kit, and the result shows that the detection of the 28 African swine fever virus positive nucleic acid samples is positive, and the detection results of the 10 negative samples and the 6 related pathogens are negative, thereby proving that the method has good specificity.
The developed kit is used for respectively detecting 8-dilution positive standard substances of the African swine fever virus for 3 times to evaluate the stability of the kit, and the results show that the 3-time detection results are consistent, which indicates that the stability of the kit is good.
Example 5 clinical application of the kit and comparison with the common fluorescent quantitative PCR method
The nucleic acids of 28 blood samples, 22 cotton swab samples and 50 tissue samples were subjected to ASFV detection by the kit of the present invention and the fluorescent quantitative PCR method recommended by the Notice of Ministry of agriculture 172 of China, and the coincidence rates of the two PCR methods were compared.
The results show that the iPCR method established in the research and the method recommended by Ministry of agriculture in China are in one-to-one correspondence to the detection of the three types of positive samples and are completely consistent. The cloning and sequencing results of 31 randomly selected positive sample samples (50%) prove that the samples are all ASFV target gene fragments, and prove that the detection effect of the kit on the African swine fever virus is superior to that of the existing fluorescence quantitative PCR method, and the kit is simpler and more portable in operation than the existing PCR method, is more suitable for field detection and diagnosis, and provides a powerful and convenient tool for the diagnosis of the African swine fever virus and epidemiological investigation.
The invention carries out freeze-drying on the reaction reagent, reduces the storage condition of the kit, can be stored for one year at the temperature of-4 ℃, and is convenient for transportation and clinical application.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest ethnic group
<120> primer, probe, kit and iPCR method for detecting African swine fever virus
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 21
<212> DNA
<213> upstream primer (ASFV)
<400> 2
cctcggcgag cgctttatca c 21
<210> 3
<211> 23
<212> DNA
<213> downstream primer (ASFV)
<400> 3
ggaaactcat tcaccaaatc ctt 23
<210> 4
<211> 15
<212> DNA
<213> Probe (ASFV)
<400> 4
cgatgcaagc tttat 15
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<212> DNA
<213> African Swine Fever Virus (ASFV)
<400> 4
cctcggcgag cgctttatca ccataagact tgcatcgcaa aaggatttgg tgaatgaatt 60
tcc 63
Claims (10)
1. A primer and a probe for detecting African swine fever virus are characterized by comprising a primer and a probe for detecting the African swine fever virus;
the primer is divided into an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. The primer and probe as claimed in claim 1, wherein the 5 'end of the nucleotide sequence of the probe is connected with a fluorescent group, and the 3' end is connected with a non-fluorescent quenching group and a quenching group;
the fluorescent group is selected from one of FAM, TET, HEX, CY3 and JOE, and is further FAM;
the quenching group is selected from one of MGB, BHQ1, BHQ2, TAMRA, DABCYL and Eclipse, and is further MGB.
3. A kit for the detection of african swine fever virus, comprising the primer and probe of claim 1 or 2.
4. The kit of claim 3, further comprising reaction buffer, Taq enzyme, reverse transcriptase, positive and negative controls, dNTPs.
5. The kit according to claim 4, characterized in that the reaction buffer consists of: 500mM KCl, 100mM Tris-HCl, 15mM MgCl2Add ddH2The total volume of O is 3ml, and the pH value is 8.0-8.5.
6. The kit according to claim 4, wherein the final concentration of Taq enzyme is 5U. mu.L-1The final concentration of reverse transcriptase was 20 U.mu.L-1。
7. The kit according to claim 4, wherein the positive control is a sample containing African swine fever virus DNA fragments;
further, the positive control is prepared by the following method: 100 mu L of DNA sample containing African swine fever virus DNA fragment is filled into a PCR tube, cooled to-40 to-80 ℃, and then freeze-dried under the air pressure of 8-12 pa to form dry powder.
8. The kit of claim 4, wherein the negative control is a sample of DNA fragments of hog cholera africana;
further, the negative control is prepared by the following method: 100 mu L of a sample of the DNA fragment of the African swine fever virus is put into a PCR tube, cooled to-40 to-80 ℃, and then lyophilized under the air pressure of 8-12 pa to form dry powder.
9. The kit according to any one of claims 3 to 8, wherein the final concentration of the primer upstream and the final concentration of the primer downstream of the African swine fever virus are both 10 μmol μ L-1The final concentration of the probe was 10. mu. mol. mu.L-1；
The final concentration of dNTPs was 2.5 mM.
10. An iPCR method for detecting African swine fever virus, which is characterized by comprising the following steps:
1) preparing a DNA template: extracting total DNA of the African swine fever virus, and preparing a DNA template;
2) establishing an iPCR reaction system:
adding 100 mu L of reaction buffer solution into the positive reference substance, mixing to prepare a positive reference substance, adding 50 mu L of reaction buffer solution and 5 mu L of positive reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube, and preparing to obtain a positive reference reaction system;
adding 100 mu L of reaction buffer solution into a negative reference substance freeze-drying tube to prepare a negative reference substance, and then adding 50 mu L of reaction buffer solution and 5 mu L of negative reference substance into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a negative reference reaction system;
adding 50 mu L of reaction buffer solution and 5 mu L of detection sample DNA into a fluorescent quantitative PCR reaction solution freeze-drying tube to prepare a detection sample DNA reaction system;
then respectively taking 50 mu L of mixture from each reaction system and transferring the mixture into a constant-temperature isolated fluorescent PCR reaction tube;
the prepared fluorescent quantitative PCR reaction tube is instantaneously centrifuged for 5s at high speed, so that bubbles are avoided;
3) and (3) amplification detection: the PCR reaction tube is placed in a constant temperature isolated fluorescent PCR instrument for reaction, and the result shows that "+" represents positive and "-" represents negative.
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