CN112760418A - Primer and kit for porcine epidemic diarrhea virus and application of primer and kit - Google Patents
Primer and kit for porcine epidemic diarrhea virus and application of primer and kit Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to a primer and a kit of porcine epidemic diarrhea virus and application thereof, wherein the primer of the porcine epidemic diarrhea virus comprises an upstream primer PEDV-MF and a downstream primer PEDV-MR, and discloses a base sequence thereof. Compared with the prior art, the primers are designed on the highly conserved E protein and M protein of PEDV in a protein spanning manner, so that the sensitivity, specificity and stability of the primers are improved.
Description
Technical Field
The invention belongs to the technical field of biology, relates to gene detection of an epidemic diarrhea virus, and particularly relates to a primer and a kit for a porcine epidemic diarrhea virus and application of the primer and the kit.
Background
PEDV belongs to the genus coronavirus, exhibits polymorphism, and has a size of 95-190nm (including fiber process). The nucleic acid is a linear single-stranded positive-stranded RNA, surrounded by a capsid. Has a capsule membrane with radial fibrous protrusions. PEDV has 4 structural proteins of spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N), and replicase 1a, 1b protein encoded by pol (polymerase) gene and ORF3 protein encoded by ORF3 gene.
Porcine Epidemic Diarrheal (PED) is an infectious disease with vomiting, diarrhea, and dehydration diarrhea as its main clinical symptoms caused by Porcine Epidemic Diarrhea Virus (PEDV). It is difficult to distinguish from Transmissible Gastroenteritis (TGE), and thus nucleic acid detection by PCR is required for confirmation.
The piglet in one week is seriously ill, part of the piglet vomits after eating and diarrhea is caused by severe dehydration death for 3-4 days, the disease course is short, the disease death rate is high and can reach 90%, and the virus propagation speed is high, so that the piglet in the stall can be screened in time after the disease occurs, isolation work is done, and the blockage of the infection source and the propagation path is very important. A large amount of detection cost is consumed in the screening process, so that a detection kit which is low in cost, rapid and accurate can be used for rapid diagnosis and timely measures.
Disclosure of Invention
The E protein and M protein of PEDV are both membrane glycoproteins of PEDV, and are key proteins for virus assembly and budding. To ensure the consistency of virus recognition to the host, both the E and M proteins are highly conserved during the genetic evolution of PED virus. According to the invention, the primers are designed on the highly conserved E protein and M protein of PEDV in a protein spanning manner, so that the sensitivity, specificity and stability of the primers are improved. Through sequence alignment, the homology of M protein and E protein of each PEDV strain is almost 100%. Therefore, primers designed on the E protein and the M protein can identify all strains of PEDV to the maximum extent.
In addition, when designing the primer of RT-PCR, the product of PCR is required to be more suitable at 500-700bp, because the RT-PCR product is too small and is easy to be confused with the primer dimer and has poor stability, the requirement of PCR product is too large for reverse transcription and the situation of mismatching is easy to occur. However, the E gene length of PEDV is 231bp, the M gene length is 681bp, the product which is singly used as the RT-PCR is smaller, and the sensitivity and the stability of the RT-PCR primer of which the amplification product is more than 300bp and designed in the M gene sequence are not ideal. According to the invention, the RT-PCR primers are designed by spanning E protein and M protein, the length of the PCR product is 669bp, and the sensitivity, specificity and stability of the primers are improved. And develops a kit convenient for diagnosis and establishes a corresponding detection method.
The E gene and the M gene in the genome of PEDV are adjacent coding regions, the E gene totally encodes 76 amino acids, the homology of the amino acid sequence at the 3' end of the E protein among different strains is almost 100%, the PEDV is an RNA virus, and the reverse transcription cDNA is used as a PCR template, so that a PCR product does not need to consider a non-coding region, and the coding regions of the E gene and the M gene can be directly spliced to be used as a template for designing a primer.
The invention aims to overcome the defects of the prior art and provide a primer and a kit of porcine epidemic diarrhea virus and application thereof.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a primer of porcine epidemic diarrhea virus, which comprises an upstream primer PEDV-MF (SEQ ID NO:1) and a downstream primer PEDV-MR (SEQ ID NO:2), and respectively has the following base sequences:
upstream primer PEDV-MF: 5'-GGGCGCGTGTATAGAGTTTA-3' the flow of the air in the air conditioner,
the downstream primer PEDV-MR: 5'-GACATAGAAAGCCCAACCAG-3' are provided.
The invention provides a kit for porcine epidemic diarrhea virus in a second aspect, which contains the primer and is marked as PCR Mix.
Preferably, the kit further comprises a positive control, a negative control, a reverse transcription reagent and a PCR amplification solution.
Preferably, in the PCR Mix, the molar ratio of the upstream primer PEDV-MF to the downstream primer PEDV-MR is 1: 1.
Preferably, the positive control is a recombinant plasmid containing the sequence of the PEDV gene.
Preferably, the negative control is double distilled water.
Preferably, the PCR amplification solution comprises Taq PCR Mix (2X).
The third aspect of the present invention provides an application of the kit for porcine epidemic diarrhea virus, which is applied to the detection of porcine epidemic diarrhea virus, and comprises the following steps:
(1) extracting total RNA of a sample to be detected, adding a reverse transcription reagent, and performing reverse transcription to obtain sample cDNA for later use;
(2) preparing a reaction system: sampling cDNA, PCR Mix and PCR amplification solution, and quantifying to a certain volume by using double distilled water to prepare a reaction system;
(3) performing RT-PCR amplification;
(4) performing agarose gel electrophoresis;
(5) processing the positive control and the negative control by referring to the sample to be detected;
(6) and (6) judging the result.
Preferably, the result determination method in step (6) is:
669bp amplified bands appear in the positive control, and the experimental result is established when no bands appear in the negative control; the 669bp amplified band of the detected sample is positive to the porcine epidemic diarrhea virus nucleic acid, otherwise, the amplified band is negative.
Preferably, in step (1), 4 μ g of RNA is taken, and reverse transcription conditions are as follows: at 42 ℃ for 45 min.
Preferably, in the reaction system of step (2), 2. mu.L of sample cDNA, 2. mu.L of PCR Mix, 12.5. mu.L of PCR amplification solution and 10.5. mu.L of double distilled water are added to quantify to 25. mu.L.
Preferably, in step (3), the amplification procedure is: 94 ℃ for 5min, cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1min, and 35 cycles in total, and 72 ℃ for 10 min.
Preferably, in step (4), the agarose gel electrophoresis method comprises: putting agarose into TAE to prepare 1% agarose gel, dissolving the agarose in microwave, adding 5 mu L nucleic acid dye, shaking up gently, putting a comb in an electrophoresis tank, pouring the agarose gel, taking 10 mu L PCR amplification product after solidification, spotting the PCR amplification product in an agarose gel hole, carrying out electrophoresis in TAE electrophoresis buffer solution at the voltage of 110-120V, observing and photographing by a gel imager after Marker and strips are separated, and obtaining a detection result.
Compared with the prior art, the invention has the following beneficial effects:
1) according to the invention, by referring to a gene sequence of a PEDV strain published in NCBI GenBank, after the highly conserved E protein and M protein of PEDV are selected for homology comparison, an RT-PCR primer is designed across the E protein and the M protein, the length of a PCR product is 669bp, and the sensitivity, specificity and stability of the primer are improved.
2) The primer sequence designed by the invention has high sensitivity, bright band and the lowest detection limit of 1.36 multiplied by 103copise/μL。
3) The kit has strong specificity, has no specific strip for porcine rotavirus, transmissible gastroenteritis virus, hog cholera virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and porcine circovirus, and ensures the detection accuracy.
4) The invention solves the problem that the sensitivity and stability of the RT-PCR primer with the amplification product more than 300bp designed in the M gene sequence are not ideal due to the short length of the M gene, establishes a kit for rapidly and accurately detecting the PEDV, has the advantages of low cost, high sensitivity, strong specificity and good stability, and is suitable for large-batch virus monitoring.
Drawings
FIG. 1 is an electrophoretogram of a band of positive control standards obtained by RT-PCR, in which M: DNA Marker; 1: negative control; 2: 669bp mesh band
FIG. 2 shows the electrophoresis results of the sensitivity test of the embodiment of the present invention, in which: m is DNA Marker; 1: negative control; 2: 1.36X 106；3:1.36×105；4:1.36×104；5:1.36×103；6:1.36×102
FIG. 3 shows the electrophoresis result of the specificity test of the embodiment of the present invention, in which: m is DNA Marker; 1: negative control; 2: porcine epidemic diarrhea virus; 3, porcine rotavirus; 4, transmissible gastroenteritis virus; 5, hog cholera virus; 6, porcine pseudorabies virus; 7: porcine reproductive and respiratory syndrome virus; 8: porcine circovirus.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Examples
1. Preparation of Positive Standard
Extracting RNA from a positive sample of known PEDV, extracting total RNA according to an RNA virus extraction kit of Tiangen, performing reverse transcription on an RNA sample template of 4 mu g to obtain total cDNA, adopting a 25 mu L system, a reaction system of 2 times RT-PCR Mix of 10 mu L, an upstream primer of 1 mu L, a downstream primer of 1 mu L, cDNA of 2 mu L, a TRUUCcript Enzyme Mix of 0.8 mu L and RNase free H2O (nuclease-free water) 10.2. mu.L; RT-PCR amplification procedure: 5min at 94 ℃; circulating at 94 ℃ for 30s, at 55 ℃ for 30s and at 72 ℃ for 1min for 35 cycles; extension was then carried out at 72 ℃ for 10 min. After amplification was complete, all products were identified by electrophoresis on a 1% agarose gel. See fig. 1. And (3) purifying and recovering the PCR product identified as positive by using a rubber recovery kit of Tiangen, connecting the PCR product to a pEASY-T1 vector, transforming the PCR product to DH5 alpha competent cells, selecting positive clones, carrying out shake bacteria amplification by using LB culture solution, and sending the bacterial solution to Shanghai biological engineering Limited company for sequencing.
Determining PCR product as target slice by sequencingSegment, total 669bp, sequence:GGGCGTTTGTATAGAGTTTA TAAGTCTTACATGCAAATAGACCCCCTCCCTAGTACTGTTATTGACGTATAAATGTCTAACGGTTCTATTCCCGTTGATGAGGTGATT CAACACCTTAGAAACTGGAATTTCACATGGAATATCATACTGACAATACTACT TGTAGTGCTTCAGTATGGCCATTACAAGTACTCTGCGTTCTTGTATGGTGTCAA GATGGCTATTCTATGGATACTTTGGCCTCTTGTGTTAGCACTGTCACTTTTTGA TGCATGGGCTAGCTTTCAGGTCAATTGGGTCTTTTTTGCTTTCAGCATCCTTAT GGCTTGCATCACTCTTATGCTGTGGATAATGTACTTTGTCAATAGCATTCGGTT GTGGCGCAGGACACATTCTTGGTGGTCTTTCAATCCTGAAACAGACGCGCTT CTCACTACTTCTGTGATGGGCCGACAGGTCTGCATTCCAGTGCTTGGAGCACC AACTGGTGTAACGCTAACACTCCTTAGTGGTACATTGCTTGTAGAGGGCTATA AGGTTGCTACTGGCGTACAGGTAAGTCAATTACCTAATTTCGTCACAGTCGCC AAGGCCACTACAACAATTGTCTACGGACGTGTTGGTCGTTCAGTCAACGCTT CATCTGGCACTGGTTGGGCTTTCTATGTC (E gene underlined and M gene not underlined).
2. Sensitivity test
Extracting standard plasmid with plasmid extraction kit of Tiangen, detecting with NanoDrop 2000 nucleic acid concentration detector to obtain a concentration of 680ng/ul, and diluting to obtain a final concentration of 1.36 × 109The copise/. mu.L was diluted 10-fold and used as a template, the concentration of which was 1.36X 106、1.36×105、1.36×104、1.36×103、1.36× 102、1.36×101、1.36×100Sensitivity determination is carried out on copise/mu L, double distilled water is used as a negative control, PCR amplification is carried out according to a reaction system and a program for providing RT-PCR, an amplification product is identified by 1% agarose gel electrophoresis, the electrophoresis result is shown in the figure, and the result shows that the 4 th lane is 1.36 multiplied by 104The copise/. mu.L band was still evident, diluted to 1.36X 103The copise/. mu.L, i.e., the band in lane 5 still has an insignificant but macroscopic band, indicates that the lowest detection limit of the present invention is 1.36X 103copise/. mu.L. As shown in FIG. 2 (in the figure: M: DNA Marker, 1: negative control; 2: 1.36X 10)6、3:1.36×105、4:1.36×104、5:1.36×103、6:1.36×102)。
3. Specificity test
The invention takes the porcine epidemic diarrhea virus, porcine rotavirus, transmissible gastroenteritis virus, hog cholera virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and porcine circovirus samples which are stored by the inventor and are identified as positive as templates, the primers are used for carrying out RT-PCR amplification, amplification products are identified by 1 percent agarose gel electrophoresis, and the electrophoresis result is shown in the figure, and the result shows that only a bright specific band appears in a PEDV lane at 669bp, and other samples do not have specific bands, which shows that the invention has stronger specificity. As shown in FIG. 3 (in the figure, M: DNA Marker; 1: negative control; 2: porcine epidemic diarrhea virus; 3: porcine rotavirus; 4: transmissible gastroenteritis virus; 5: swine fever virus; 6: porcine pseudorabies virus; 7: porcine reproductive and respiratory syndrome virus; 8: porcine circovirus).
In the sensitivity test and specificity test processes, the reaction system and the procedure of RT-PCR are as follows:
the kit of the porcine epidemic diarrhea virus comprises a PCR Mix (primer), a positive control, a negative control, a reverse transcription reagent and a PCR amplification solution.
In PCR Mix:
upstream primer PEDV-MF: 5'-GGGCGCGTGTATAGAGTTTA-3' the flow of the air in the air conditioner,
the downstream primer PEDV-MR: 5'-GACATAGAAAGCCCAACCAG-3' are provided.
The molar ratio of the upstream primer PEDV-MF to the downstream primer PEDV-MR is 1: 1.
The positive control is a recombinant plasmid containing the sequence of the PEDV gene.
The negative control was double distilled water.
PCR amplification solution included Taq PCR Mix (2X).
When in detection, the method comprises the following steps:
(1) extracting total RNA of a sample to be detected, taking 4 mu g of RNA, adding a reverse transcription reagent, carrying out reverse transcription at 42 ℃ for 45min to obtain sample cDNA for later use;
(2) preparing a reaction system:
2 mu L of sample cDNA, 2 mu L of PCR Mix and 12.5 mu L of PCR amplification solution, and adding 10.5 mu L of double distilled water for quantifying to 25 mu L to prepare a reaction system;
(3) and (3) RT-PCR amplification:
the amplification procedure was: at 94 ℃ for 5min, the cycle is 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1min, and the total number of the cycles is 35 and the temperature is 72 ℃ for 10 min;
(4) agarose gel electrophoresis:
putting agarose into TAE to prepare 1% agarose gel, dissolving the agarose in microwave, adding 5 mu L nucleic acid dye, shaking up lightly, putting a comb in an electrophoresis tank, pouring the agarose gel, taking 10 mu L PCR amplification product after solidification, spotting the PCR amplification product in an agarose gel hole, carrying out electrophoresis in TAE electrophoresis buffer solution at the voltage of 110-120V, observing and photographing by a gel imager after a Marker and a strip are separated, and obtaining a detection result;
(5) processing the positive control and the negative control by referring to the sample to be detected;
(6) and (4) judging a result:
669bp amplified bands appear in the positive control, and the experimental result is established when no bands appear in the negative control; the 669bp amplified band of the detected sample is positive to the porcine epidemic diarrhea virus nucleic acid, otherwise, the amplified band is negative.
The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> Fujian Aaonong Biotechnology group Ltd
ZHANGPU COUNTY ZHAOMULAN BREEDING Co.,Ltd.
XIAMEN JIAYEXING AGRICULTURAL TECHNOLOGY Co.,Ltd.
Fujian Hake ecological agriculture Co Ltd
Primer and kit for porcine epidemic diarrhea virus and application of primer and kit
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gacatagaaa gcccaaccag 20
Claims (10)
1. The primers for the porcine epidemic diarrhea virus are characterized by comprising an upstream primer PEDV-MF and a downstream primer PEDV-MR which respectively have the following base sequences:
upstream primer PEDV-MF: 5'-GGGCGCGTGTATAGAGTTTA-3' the flow of the air in the air conditioner,
the downstream primer PEDV-MR: 5'-GACATAGAAAGCCCAACCAG-3' are provided.
2. A kit for porcine epidemic diarrhea virus, comprising the primers of claim 1 and labeled as PCR Mix.
3. The kit of claim 2, further comprising a positive control, a negative control, a reverse transcription reagent, and a PCR amplification solution.
4. The kit for porcine epidemic diarrhea virus of claim 2 or 3, wherein the molar ratio of the upstream primer PEDV-MF to the downstream primer PEDV-MR in the PCR Mix is 1: 1.
5. The kit according to claim 3, wherein the positive control is a recombinant plasmid containing the PEDV gene sequence.
6. The kit for porcine epidemic diarrhea virus of claim 3, wherein the negative control is double distilled water.
7. The kit of claim 3, wherein the PCR amplification solution comprises Taq PCR Mix (2X).
8. The use of the kit according to any one of claims 2 to 7 for the detection of porcine epidemic diarrhea virus, comprising the steps of:
(1) extracting total RNA of a sample to be detected, adding a reverse transcription reagent, and performing reverse transcription to obtain sample cDNA for later use;
(2) preparing a reaction system: sampling cDNA, PCR Mix and PCR amplification solution, and quantifying to a certain volume by using double distilled water to prepare a reaction system;
(3) performing RT-PCR amplification;
(4) performing agarose gel electrophoresis;
(5) processing the positive control and the negative control by referring to the sample to be detected;
(6) and (6) judging the result.
9. The use of the kit for porcine epidemic diarrhea virus according to claim 8, wherein the method for determining the result in step (6) comprises:
669bp amplified bands appear in the positive control, and the experimental result is established when no bands appear in the negative control; the 669bp amplified band of the detected sample is positive to the porcine epidemic diarrhea virus nucleic acid, otherwise, the amplified band is negative.
10. Use of a kit of porcine epidemic diarrhea virus according to claim 8, characterized in that it comprises any one or more of the following conditions:
(i) in the step (1), 4 mu g of RNA is taken, and reverse transcription conditions are as follows: at 42 deg.C for 45 min;
(ii) in the reaction system of the step (2), 2 mu L of sample cDNA, 2 mu L of PCR Mix and 12.5 mu L of PCR amplification solution are added with 10.5 mu L of double distilled water to quantify to 25 mu L;
(iii) in the step (3), the amplification procedure is as follows: at 94 ℃ for 5min, the cycle is 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1min, and the total number of the cycles is 35 and the temperature is 72 ℃ for 10 min;
(iv) in the step (4), the agarose gel electrophoresis method comprises the following steps: putting agarose into TAE to prepare 1% agarose gel, dissolving the agarose in microwave, adding 5 mu L nucleic acid dye, shaking up gently, putting a comb in an electrophoresis tank, pouring the agarose gel, taking 10 mu L PCR amplification product after solidification, spotting the PCR amplification product in an agarose gel hole, carrying out electrophoresis in TAE electrophoresis buffer solution at the voltage of 110-120V, observing and photographing by a gel imager after Marker and strips are separated, and obtaining a detection result.
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