CN114317815A - PCR method for amplifying N-terminal hypervariable region of S gene of porcine epidemic diarrhea virus - Google Patents
PCR method for amplifying N-terminal hypervariable region of S gene of porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention relates to the technical field of animal virology and animal infectious disease, and discloses a primer for amplifying PCR (polymerase chain reaction) of a high mutation region at the N tail end of an S gene of porcine epidemic diarrhea virus. The method further provides the whole process of the treatment of the clinical pig diarrhea feces and intestinal tissue samples, the extraction of total RNA, the reverse transcription, the PCR, the electrophoresis gel-separation and the result judgment. Using this method, a 1823bp fragment was seen for positive samples. The amplification primer provided by the invention has good conservation, the PCR method has good specificity and simple and rapid operation, is convenient for clinically detecting the porcine epidemic diarrhea infection, and can analyze the subsequent sequence of the obtained product for identifying the strain type.
Description
Technical Field
The invention relates to the technical field of animal virology and animal infectious disease science, in particular to a PCR method for amplifying a high mutation region at the N tail end of an S gene of porcine epidemic diarrhea virus.
Background
Porcine Epidemic Diarrhea (PED) is an acute, highly transmitted disease, the main etiology of which is Porcine Epidemic Diarrhea Virus (PEDV). The disease is mainly characterized clinically by vomiting, watery diarrhea, dehydration and the like, and pigs at all ages are susceptible to the disease. But has strong pathogenicity to the suckling piglets, the morbidity is 100 percent, and the mortality rate is up to 80 to 100 percent. PED genus alpha-coronavirus, which is a enveloped, non-segmented, single-stranded positive-strand RNA virus, has a genome length of about 28kb and comprises a 5 'cap structure and a 3' polyA tail. The whole genome of PEDV consists of a 5 'end non-coding region, a 3' UTR and at least 7 open reading frames (Openreadingfram, ORF), which encode 3 non-structural proteins (replicase 1a, 1b and non-structural protein ORF3) and 4 structural proteins (S, E, M and N), which are in the genome in the order 5 'UTR-P1 a/1b-S-ORF 3-E-M-N-3' UTR.
The epidemic situation is caused by the variant PEDV strain, animal experiments prove that the PEDV variant strain has stronger pathogenicity, and the traditional vaccine prepared by the classical strain cannot provide effective immunoprotection. Compared with classical strains, PEDV variant strains have variant characteristics mainly focused on the S gene.
The PEDVS gene, one of the most important structural proteins of the virus, plays an important role in virus invasion and induction of neutralizing antibodies. The S protein can be divided into two functional regions of S1 (amino acids 1-789) and S2 (amino acids 790-1383): wherein S1 contains the virus key neutralizing epitope (COE) and receptor binding domain, S2 is primarily responsible for the membrane fusion process of the virus with the host. The homology between the S genes of different coronaviruses is low; within PEDV, the variability of the S gene between different strains is also large. The S gene is also the gene which is most prone to variation in each protein gene of the PEDV virus, and homology analysis aiming at the S gene is also a key method for distinguishing virus subtypes.
Previous studies have found that PEDV mainly has 4 variant regions V1, V2, V3 and V4, wherein V2 is located in the 20,661 to 22,300 base region (refer to PEDV variant reference strain AH2012), and the region is mainly concentrated at the N-terminal of S gene, and the region has the most important structure-COE for inducing neutralizing antibody, so that it is important for accurate and rapid amplification of the region and subsequent sequencing analysis to diagnose PEDV and determine its infection type in clinic.
Therefore, through whole genome sequence alignment analysis of a plurality of different PEDV strains, the applicant designs and synthesizes a pair of PCR primers for amplifying the N-terminal hypervariable region-V2 of the PEDVS gene at the conserved position of the gene sequence containing the PEDVV2 region, establishes a reverse transcription PCR method, and can be used for obtaining the type analysis products of the PEDV infection strains in clinic.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a PCR method for amplifying a hypervariable region at the N terminal of an S gene of porcine epidemic diarrhea virus, and solves the problems.
(II) technical scheme
In order to achieve the above purpose, the invention provides the following technical scheme: the sequences of the upstream primer and the downstream primer of the PCR method for amplifying the high mutation region at the N tail end of the S gene of the porcine epidemic diarrhea virus are SEQ ID NO.1 and SEQ ID NO. 2:
upstream primer PEDV-V2-F: 5'-TGGTAAGTTGCTAGTGCGTAATAAT-3'
The downstream primer PEDV-V2-R: 5'-AAAGACAGGTAATCATTAACAGATT-3'.
The PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus comprises the following steps:
(1) extracting clinical pig diarrhea pathological material RNA: collecting 100mg of loose stool or 100mg of animal tissue, cutting into pieces with tissue scissors, adding 350 μ l TRK lysate in RNA extraction kit of OMEGA company, mixing, and standing at room temperature for 5 min; centrifuging at 12000rpm/min for 5min, sucking 350 μ l of supernatant, adding 250 μ l of anhydrous ethanol, mixing, and standing at room temperature for 5 min; transferring all the above liquid onto HibindRNAMINicolumn, centrifuging at 10,000g room temperature for 1min, and discarding the waste liquid; placing the column in a new collection tube of 2mL, adding 500. mu.l of RNAwashbuffer I, centrifuging at 10,000g for 1min at room temperature, and discarding the waste liquid; placing the column in a 2mL collection tube, adding 500. mu.l of RNAwashbuffer II (diluted with ethanol), centrifuging at 10,000g for 30s at room temperature, and discarding the waste liquid; repeating the above operation once; the RNA binding column was then placed in an empty collection tube and centrifuged at 13,000g for 2min at room temperature to completely dry the Hibind matrix; transferring the column to a new 1.5ml Ep tube, eluting the column with 50. mu.l of DEPC water, and centrifuging at 13,000g for 2min at room temperature, wherein the collected liquid is total RNA extracted;
(2) reverse transcription of total RNA: RNA was reverse transcribed into cDNA using OligodT (18) as a reverse transcription primer, according to the instructions of SuperScriptTMIIIReverseTranscriptase product of Invitrogen.
The template system is as follows: RNA template 12. mu.l, 10mM dNTPs 1. mu.l, downstream primer 1. mu.l total volume 14. mu.l, after mixing, water bath at 65 ℃ for 5min, then placing in ice bath for at least 1 min. Then preparing a reaction system: 5 XFirst-strandbuffer 4. mu.l, 0.1MDTT 1. mu.l, reverse transcriptase 1. mu.l, total volume 6. mu.l.
Mixing the two systems, placing in 55 deg.C water bath for 1h, inactivating in 70 deg.C water bath for 15min after reaction, ice-cooling for 3min, and storing the synthesized cDNA at-20 deg.C.
(3) Adding the upstream and downstream primers and the PCR mixed solution into a PCR reaction tube, and adding cDNA of a sample to be detected after mixing;
(3) placing the PCR tube in a PCR amplification instrument for circulation;
(4) taking the PCR product to perform electrophoresis on 1% agarose gel;
and (4) judging a result: the amplified product is a 1823bp band which is positive; no amplification product was negative at approximately 1823 bp.
The PCR mixture of step (2) comprises: using a 25. mu.l PCR reaction system, 2.5. mu.l of 10 XPCR buffer, 1.0. mu.l of dNTP (2.5mM), 1.5. mu.l of MgCl2(25mM), 0.5. mu.l of each of the upstream and downstream primers, 0.5. mu.l of 5U/. mu.l of TaqDNA polymerase, and 1. mu.l of DNA template were put into a 0.2ml PCR reaction tube, and then sterilized ultrapure water was added to the total volume and mixed. The amplification conditions of the step (3) are as follows: performing pre-denaturation at 94 ℃ for 5 min; then circulating for 35 times at 94 ℃,30s, 55 ℃,30s, 72 ℃ and 90 s; finally, extension is carried out for 10min at 72 ℃.
The PCR method is used for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus, the positive reference substance is a standard positive plasmid containing the PEDV-V2 gene, and the concentration is 1.5 multiplied by 1011 copies/mu l.
(III) advantageous effects
Compared with the prior art, the PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus has the following beneficial effects:
1. according to the PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus, the invention selects conserved regions at two ends including the hypervariable region V2 to design and synthesize a pair of specific primers according to the genome sequences of a plurality of porcine PEDV classical strains and variant strains published in GenBank, and performs PCR amplification on a porcine PEDV positive sample to obtain a 1823bp specific band.
2. The PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus is characterized in that the PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus, which is established by the research, is used for other common porcine coronaviruses: porcine transmissible gastroenteritis virus, porcine delta coronavirus, and other common porcine pathogens: the porcine reproductive and respiratory syndrome virus, the porcine circovirus type 2, the classical swine fever virus and the pseudorabies virus have no response.
3. According to the PCR method for amplifying the N-terminal hypervariable region of the S gene of the porcine epidemic diarrhea virus, through aiming at the feces and intestinal tissue samples of clinically infected pigs with PEDV, DNA fragments with the size of about 1823bp are amplified by the PCR method established by the invention, the PCR method is suitable for detecting clinical pathogeny, and the obtained amplified fragments can be used for sequencing analysis of types of PEDV infected strains.
Drawings
FIG. 1 is a schematic diagram of the design of primers for PCR method for amplifying the N-terminal hypervariable region of S gene of porcine epidemic diarrhea virus;
FIG. 2 is a diagram showing the result of electrophoresis of a PCR method specific test for detecting the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to FIGS. 1-2, a PCR method for amplifying an N-terminal hypervariable region of an S gene of porcine epidemic diarrhea virus, comprises the steps of:
(1) extracting clinical pig diarrhea pathological material RNA: collecting 100mg of loose stool or 100mg of animal tissue, cutting into pieces with tissue scissors, adding 350 μ l TRK lysate in RNA extraction kit of OMEGA company, mixing, and standing at room temperature for 5 min; centrifuging at 12000rpm/min for 5min, sucking 350 μ l of supernatant, adding 250 μ l of anhydrous ethanol, mixing, and standing at room temperature for 5 min; transferring all the above liquid onto HibindRNAMINicolumn, centrifuging at 10,000g room temperature for 1min, and discarding the waste liquid; placing the column in a new collection tube of 2mL, adding 500. mu.l of RNAwashbuffer I, centrifuging at 10,000g for 1min at room temperature, and discarding the waste liquid; placing the column in a 2mL collection tube, adding 500. mu.l of RNAwashbuffer II (diluted with ethanol), centrifuging at 10,000g for 30s at room temperature, and discarding the waste liquid; repeating the above operation once; the RNA binding column was then placed in an empty collection tube and centrifuged at 13,000g for 2min at room temperature to completely dry the Hibind matrix; transferring the column to a new 1.5ml Ep tube, eluting the column with 50. mu.l of DEPC water, and centrifuging at 13,000g for 2min at room temperature, wherein the collected liquid is total RNA extracted;
(2) reverse transcription of total RNA: RNA was reverse transcribed into cDNA using OligodT (18) as a reverse transcription primer, according to the instructions of SuperScriptTMIIIReverseTranscriptase product of Invitrogen. The template system is as follows: RNA template 12. mu.l, 10mM dNTPs 1. mu.l, downstream primer 1. mu.l total volume 14. mu.l, after mixing, water bath at 65 ℃ for 5min, then placing in ice bath for at least 1 min. Then preparing a reaction system: 5 XFirst-strandbuffer 4. mu.l, 0.1MDTT 1. mu.l, reverse transcriptase 1. mu.l, total volume 6. mu.l. Mixing the two systems, placing in 55 deg.C water bath for 1h, inactivating in 70 deg.C water bath for 15min after reaction, ice-cooling for 3min, and storing the synthesized cDNA at-20 deg.C.
(3) Using a 25. mu.l PCR reaction system, 2.5. mu.l of 10 XPCR buffer, 1.0. mu.l of dNTP (2.5mM), 1.5. mu.l of MgCl2(25mM), 0.5. mu.l of each of the upstream and downstream primers, 0.5. mu.l of 5U/. mu.l of TaqDNA polymerase, and 1. mu.l of DNA template were put into a 0.2ml PCR reaction tube, and then sterilized ultrapure water was added to the total volume and mixed.
(4) The PCR amplification conditions were: performing pre-denaturation at 94 ℃ for 5 min; then circulating for 35 times at 94 ℃,30s, 56 ℃,30s, 72 ℃ and 1 min; finally, extension is carried out for 10min at 72 ℃.
(4) 10 mul of the PCR product is electrophoresed on 1% agarose gel, and the result shows that the amplification product of the loose stool and the intestinal tissue is about 1823bp, the size is consistent, and the product is judged to be PEDV positive.
Example 2
As in example 1, with porcine PEDV positive disease as a positive control, the treatment of other common porcine coronaviruses in pigs: porcine transmissible gastroenteritis virus, porcine delta coronavirus, and other common porcine pathogens: the results of the detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, classical swine fever virus and pseudorabies virus fail to amplify any band, and the amplification of porcine PEDV positive disease material generates a band of about 1823bp, which is shown in figure 1. The sequence is SEQ ID NO. 3:
according to figure 2, lane 1 is a negative control and lane 2 is a porcine PEDV positive sample; lanes 3-8 are: porcine transmissible gastroenteritis virus, porcine delta coronavirus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, classical swine fever virus, pseudorabies virus; m represents DNAmarker (DL 2000).
The primer sequence is as follows:
SEQIDNO.1：5’-TGGTAAGTTGCTAGTGCGTAATAAT-3’
SEQIDNO.2：5’-AAAGACAGGTAATCATTAACAGATT-3’
amplifying a reference sequence of the target gene:
SEQ ID No. 3: reference strain AH2012, 20566-22388bp region of PEDV variant
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A primer for amplifying PCR of a hypervariable region at the N terminal of an S gene of porcine epidemic diarrhea virus comprises an upstream primer and a downstream primer, and is characterized in that: the sequences of the upstream primer and the downstream primer are SEQ ID NO.1 and SEQ ID NO.2 respectively.
2. The primers for PCR amplification of the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus of claim 1, wherein: the upstream primer PEDV-V2-F is 5'-TGGTAAGTTGCTAGTGCGTAATAAT-3';
the downstream primer PEDV-V2-R: 5'-AAAGACAGGTAATCATTAACAGATT-3'.
3. A PCR method for amplifying a hypervariable region at the N-terminal end of the S gene of porcine epidemic diarrhea virus, comprising the steps of:
(1) extracting clinical pig diarrhea pathological material RNA: cutting 100mg of feces or 100mg of animal tissue with tissue scissors, mixing 350. mu.l of TRK lysate in RNA extraction kit, standing at room temperature for 5min, centrifuging at 12000rpm/min for 5min, sucking 350. mu.l of supernatant, adding 250. mu.l of absolute ethanol, mixing, standing at room temperature for 5min, transferring all the above-mentioned liquid to HibindRNAM, centrifuging at room temperature for 1min, discarding waste liquid, placing the column in a new 2mL collection tube, adding 500. mu.l of RNAwashbuffer I, centrifuging at room temperature for 1min, discarding waste liquid, placing the column in 2mL collection tube, adding 500. mu.l of RNAwashbuffer II (diluted with ethanol), centrifuging at room temperature for 30s, discarding waste liquid, repeating the above operations once, placing the RNA-bound column in an empty collection tube, centrifuging at room temperature for 2min, 13,000g of RNAwashbuffer II (diluted with ethanol), transferring the column to a new Ep1.5. mu.l PC (elution column) with 50. mu.l of water, centrifuging at room temperature for 2min at 13,000g, wherein the collected liquid is total extracted RNA;
(2) reverse transcription of total RNA, namely reverse transcription of the total RNA of a sample to obtain a cDNA template;
(3) adding the upstream and downstream primers and the PCR mixed solution into a PCR reaction tube, and adding cDNA of a sample to be detected after mixing;
(4) placing the PCR tube in a PCR amplification instrument for circulation;
(5) taking the PCR product to perform electrophoresis on agarose gel with the mass ratio of 1%;
(6) and (4) judging a result: the amplified product is about 1823bp band and is positive, namely PEDV exists, and the non-amplified product is about 1823bp band and is negative, namely PEDV does not exist.
4. The PCR method for amplifying the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus of claim 3, wherein the PCR mixture of step (2) comprises: using 25. mu.l of a PCR reaction system, 2.5. mu.l of 10 XPCR buffer, 1.0. mu.l of 2.5mM dNTP, 21.5. mu.l of 25mM MgCl, 0.5. mu.l of each of the upstream and downstream primers, 0.5. mu.l of 5U/. mu.l TaqDNA polymerase, and 1. mu.l of a DNA template were put into a 0.2ml PCR reaction tube, added to the total volume with sterilized ultrapure water, and mixed.
5. The PCR method for amplifying the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus of claim 3, wherein the amplification conditions of step (3) are as follows: pre-denaturation at 94 ℃ for 5min, followed by 35 cycles at 94 ℃,30s, 55 ℃,30s, 72 ℃,90s, and final extension at 72 ℃ for 10 min.
6. The PCR method for amplifying the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus of claim 3, wherein the positive control is a standard positive plasmid containing the PEDV-V2 gene and the concentration is 1.5 x 1011copies/μ l.
7. The PCR method for amplifying the N-terminal hypervariable region of the S gene of porcine epidemic diarrhea virus of claim 3, wherein the amplification product is about 1823bp and has the nucleotide sequence of SEQ ID NO. 3.
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