CN111440902A - Porcine epidemic diarrhea virus detection primer, kit and application thereof - Google Patents
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Abstract
The invention provides a porcine epidemic diarrhea virus detection primer, a kit and application thereof. The invention establishes a DPO-RT-PCR detection method aiming at Porcine Epidemic Diarrhea Virus (PEDV) and a corresponding detection kit by introducing a double Priming Oligonucleotide primer (DPO) design technology, designing a DPO primer according to a highly conserved specific sequence of the PEDV and optimizing a reaction system. The kit has the characteristics of rapidness, convenience, strong specificity, high sensitivity and good reliability, can realize accurate detection on PEDV and assist prevention, control, diagnosis and treatment of the PEDV, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of animal epidemic disease detection, and particularly relates to a porcine epidemic diarrhea virus detection primer, a kit and application thereof.
Background
Porcine Epidemic Diarrhea (PED) was first developed in the United kingdom in 1971, and the onset of disease in China was successively reported in the beginning of the 80 th 20 th century. Porcine epidemic diarrhea, commonly known as winter diarrhea, occurs in the form of outbreaks in non-immune weaned piglets (type I) or pigs of various ages (type II), is an acute intestinal infectious disease of pigs caused by Porcine Epidemic Diarrhea Virus (PEDV), and can cause the pigs to have symptoms such as watery diarrhea, vomiting, dehydration, and the like.
PEDV belongs to the genus coronaviridae (Coronavirinae) belonging to the family coronaviridae (Coronavirus), and its genome is an infectious single-stranded positive-strand RNA virus, has a total length of about 28kb, and mainly includes 6 ORFs, and genes encoding replicase-encoding polyprotein 1ab (pp1ab), spike protein (S), ORF protein, small membrane protein (E), membrane glycoprotein (M), and nucleocapsid protein (N) are present in this order from 5 'to 3'. To date, no different serotypes of PEDV have been found. PEDV is sensitive to ether, chloroform. The clinical change and symptoms of the porcine epidemic diarrhea are very similar to those of the porcine transmissible gastroenteritis, and the porcine epidemic diarrhea and the porcine transmissible gastroenteritis have no obvious difference in terms of epidemiology and clinical symptoms, so the diagnosis is difficult and needs to be diagnosed in a laboratory.
At present, the diagnosis methods of domestic laboratories comprise pathology examination, serological detection, RT-PCR detection and the like, but the existing detection methods have limitations, are easily limited by factors such as detection conditions, periods, accuracy and the like, and are difficult to realize efficient and rapid pathogen diagnosis, such as virus separation and serological detection methods, which are time-consuming and labor-consuming, low in sensitivity and easy to generate false positive, while the conventional RT-PCR method has a complex primer design process, primer parameters need to be repeatedly optimized, detection sensitivity and specificity need to be improved, and the occurrence of non-specific amplification is difficult to avoid. The DPO double-start oligonucleotide primer (DPO) is a novel primer design method, the specificity of the primer is higher than that of a conventional primer, more than 3 basic groups of mismatch occur between the DPO primer and template DNA, the amplification efficiency can be greatly reduced or the reaction can be terminated, meanwhile, the effective annealing temperature range of the DPO primer is wide, the design process is simple, and the complicated steps of conventional primer design are greatly simplified.
In view of the above, the present invention is expected to establish a DPO-RT-PCR method for detecting PEDV by introducing a Dual Priming Oligonucleotide (DPO) design technology, so as to improve the sensitivity and specificity of detection and realize accurate detection of PEDV.
Disclosure of Invention
The invention provides a porcine epidemic diarrhea virus detection primer, a kit and application thereof, wherein a double-start Oligonucleotide primer (DPO) design technology is introduced, gene sequences of PEDV are compared, a high-specificity sequence of the PEDV is selected for designing a DPO primer, and a high-sensitivity high-specificity detection method for the PEDV is established, so that the precise detection of the PEDV is realized, and the prevention, control, diagnosis and treatment of the PEDV are assisted.
To this end, the first aspect of the present invention provides a primer for detecting porcine epidemic diarrhea virus, wherein the primer comprises:
an upstream primer: 5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3' (SEQ ID NO.1),
a downstream primer: 5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3' (SEQ ID NO.2),
wherein I represents inosine.
The invention provides a porcine epidemic diarrhea virus detection kit in a second aspect, which comprises upstream and downstream primers shown in SEQ ID No.1 and SEQ ID No.2, and also comprises a positive quality control product and a negative quality control product.
In an embodiment of the invention, in the kit for detecting porcine epidemic diarrhea virus, the final reaction concentration of the upstream primer and the downstream primer in a reaction system is 0.025-0.6 μ M.
In a preferred embodiment of the invention, the final concentration of the upstream and downstream primer reaction in the reaction system of the porcine epidemic diarrhea virus detection kit is 0.2-0.6 μ M.
In an embodiment of the invention, the porcine epidemic diarrhea virus detection kit has reaction conditions including:
in an embodiment of the invention, the porcine epidemic diarrhea virus detection kit has reaction conditions including:
in an embodiment of the invention, the detection sensitivity of the porcine epidemic diarrhea virus detection kit can reach 6.84 × 103copies/μL。
In an embodiment of the present invention, the porcine epidemic diarrhea virus detection kit can specifically identify the porcine epidemic diarrhea virus in a sample, wherein the sample comprises one or more of porcine transmissible gastroenteritis virus, porcine acute diarrhea syndrome coronavirus, porcine delta coronavirus, porcine rotavirus, porcine respiratory coronavirus disease and porcine semaglaia valley virus.
The third aspect of the invention provides a method for detecting porcine epidemic diarrhea virus, which comprises the following steps:
1) extracting nucleic acid from a sample to be detected;
2) performing RT-PCR amplification by using the nucleic acid obtained in the step 1) as an amplification template and using the primer of the first aspect of the invention to obtain an amplification product;
3) and carrying out agarose gel electrophoresis on the amplification product to obtain a detection result.
In one embodiment of the present invention, the agarose gel of step 3) is a 1% agarose gel.
In a fourth aspect, the invention provides a primer for detecting porcine epidemic diarrhea virus according to the first aspect, a kit for detecting porcine epidemic diarrhea virus according to the second aspect, or a method for detecting porcine epidemic diarrhea virus according to the third aspect.
The kit provided by the invention has the advantages of rapidness, simplicity, convenience, strong specificity, high sensitivity, good reliability, low detection cost, low level requirement on detection instruments and operators, high detection sensitivity and specificity and capability of accurately identifying and diagnosing the trace latent infection of the porcine epidemic diarrhea virus, so that the kit is suitable for monitoring and early warning of the trace latent infection period of the porcine epidemic diarrhea virus, has good application value and is beneficial to application and popularization in breeding enterprises.
Drawings
Fig. 1 is a primer annealing temperature optimization result of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, where M is DNAMarker1000, and 1 to 6 are: 42 ℃, 46 ℃, 50 ℃, 54 ℃, 58 ℃ and 62 ℃;
FIG. 2 shows the result of optimizing the primer concentration of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAmarker1000, and 1-8 are: 0.025. mu.M, 0.05. mu.M, 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M, 0.6. mu.M;
fig. 3 is a specific test result of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, where M is DNAMarker1000, 1 is a positive control, and 2 to 7 are: porcine transmissible gastroenteritis virus (TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), porcine rotavirus (PoRV), porcine respiratory coronavirus disease (PRCV) and porcine intrastoppered valley virus (SVV) samples, and 8 is a negative control;
FIG. 4 shows the sensitivity test results of the porcine epidemic diarrhea virus DPO-RT-PCR method provided in the embodiment of the present invention, wherein M is DNAmarker1000, and 1-8 are 10-fold samples diluted in gradient, and the dilution is sequentially: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8；
Fig. 5 is a detection result of the porcine epidemic diarrhea virus DPO-RT-PCR method on a clinical sample provided in an embodiment of the present invention, where M is DNAMarker1000, 1 is a negative control, 2 is a positive control, and 3-24 are clinical samples.
Detailed Description
While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions. In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.
Example 1 construction of RT-PCR Rapid detection method
1. Designing a primer:
based on the gene sequences of Porcine Epidemic Diarrhea Virus (PEDV) registered in GenBank, N gene was selected as a primer design region using oligo6.0 primer design software, and the primer sequences were as follows:
an upstream primer PEDV-DPO-F:
5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3'(SEQIDNO:1)
the downstream primer PEDV-DPO-R:
5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3'(SEQIDNO:2)
2. extraction of sample RNA:
a humoral virus DNA/RNA preparation kit (cat. No.: PID0320425) from Kangning Life sciences (Wujiang) Co., Ltd.) was used to extract template RNA according to the instructions and stored at-70 ℃ for future use.
3. RT-PCR reaction:
RT-PCR was performed using a one-step RT-PCR kit (RR057A) from Baozi physician's technology (Beijing) Co., Ltd., using the sample RNA extracted in step 2 as a template RNA according to the instructions. The 25. mu.l reaction was as follows:
the reaction procedure was as follows:
4. and (3) judging a detection result:
and (3) identifying the RT-PCR product through 1% agarose gel electrophoresis, and if a single target band with the size of 361bp can be amplified, judging that the sample is positive to the porcine epidemic diarrhea virus.
5. Gene cloning, construction of plasmid containing target gene
And (3) recovering a PCR product by using an agarose gel DNA recovery kit, connecting the PCR product with a PMD-19T vector overnight, and then transforming escherichia coli DH5 α.
6. Sequencing identification
And carrying out PCR and double enzyme digestion identification on the obtained plasmid, sending the plasmid containing the target gene to Dahuanong biotechnology limited company of West, Guangdong for sequencing, and carrying out comparison and confirmation to obtain the positive plasmid.
7. DPO-RT-PCR condition optimization
PCR amplification was performed on a gradient PCR instrument for different annealing temperatures (42 ℃, 46 ℃, 50 ℃, 54 ℃, 58 ℃ and 62 ℃) and primer concentrations (0.025. mu.M, 0.05. mu.M, 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M and 0.6. mu.M), and the optimal reaction conditions for PCR were investigated. The annealing temperature optimization result is shown in FIG. 1, and the result shows that the used annealing temperatures can amplify target bands, which indicates that the effective annealing temperature range of the DPO primer is wide. The optimized primer concentration result is shown in figure 2, the effective concentration of the DPO primer is 0.025-0.6 mu M, and when the concentration of the DPO primer is 0.2-0.6 mu M, the PCR amplification effect is better.
8. Specificity of the DPO-RT-PCR method
According to the RT-PCR reaction system and the reaction program in the step 3, the sample template RNA is respectively changed into a porcine transmissible gastroenteritis virus (TGEV), a porcine acute diarrhea syndrome coronavirus (SADS-CoV), a porcine delta coronavirus (PDCoV), a porcine rotavirus (PoRV), a porcine respiratory coronavirus disease (PRCV) and a porcine Severe Valley Virus (SVV) sample, the porcine epidemic diarrhea virus positive sample detected and identified by sequencing in the laboratory is used as a positive control, the RT-PCR reaction is carried out, the RT-PCR product is identified by 1% agarose gel electrophoresis, the electrophoresis result is shown in figure 3, the result shows that only a single band appears at 361bp of the positive control lane, and the method has good specificity.
9. Sensitivity of the DPO-RT-PCR method
The RT-PCR reaction system and reaction procedure described in step 3 were followed to obtain RNA (6.84 × 10) as a positive sample in step 89copies/mu L) as template according to 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8Performing 10-fold gradient dilution, performing RT-PCR reaction with deionized water as negative control, identifying RT-PCR product by 1% agarose gel electrophoresis, and showing that the dilution is 10-5The bands were still evident, dilution 10-6The band still remained indistinct but visible to the naked eye, indicating 10-6For the lowest detection limit, the single-item DPO-RT-PCR of PEDV has the lowest detection of 6.84 × 103copies/μL。
10. Clinical sample testing
And (3) according to the RT-PCR reaction system and the reaction program in the step (3), detecting 22 clinical samples suspected of generating the porcine epidemic diarrhea virus by taking the positive control RNA in the step (8) as a positive control and deionized water as a negative control, extracting template RNA and carrying out RT-PCR reaction according to the steps (2) and (3), identifying RT-PCR products by 1% agarose gel electrophoresis, and obtaining an electrophoresis result shown in figure 5. The results showed that the negative-positive control was established and 7 samples were positive for PEDV.
In conclusion, the kit for detecting the porcine epidemic diarrhea virus provided by the invention has higher detection sensitivity and specificity, has lower requirements on equipment and personnel skills, is suitable for monitoring and early warning of the trace recessive infection period of the porcine epidemic diarrhea virus, is beneficial to application and popularization in breeding enterprises, can greatly increase the pathogen detection rate under the condition of trace infection of the porcine epidemic diarrhea virus, can accurately identify and diagnose the cause of the porcine infection, is beneficial to applying a correct treatment scheme for symptomatic medication, and has a good application prospect.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> porcine epidemic diarrhea virus detection primer, kit and application thereof
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>31
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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ggtgagcgaa ttgaacaacc ttcggcattt c 31
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<213> Artificial Sequence (Artificial Sequence)
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gaccctggtt atttccacga ttctttacca c 31
Claims (8)
1. The primers for detecting the porcine epidemic diarrhea virus are characterized by comprising the following components:
an upstream primer: 5'-GGTGAGCGAATTGAACAACCTTCIIIIIGGCATTTC-3' (SEQ ID NO.1),
a downstream primer: 5'-GACCCTGGTTATTTCCACGATTCTIIIIITTACCAC-3' (SEQ ID NO.2),
wherein I represents inosine.
2. A porcine epidemic diarrhea virus detection kit is characterized by comprising upstream and downstream primers shown in SEQ ID No.1 and SEQ ID No.2, and further comprising a positive quality control substance and a negative quality control substance.
3. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the final concentration of the upstream and downstream primer reactions in the reaction system of the kit is 0.025-0.6 μ M.
4. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit reaction conditions comprise:
5. the porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit has a detection sensitivity of up to 6.84 × 103copies/μL。
6. The porcine epidemic diarrhea virus detection kit of claim 2, wherein the kit specifically identifies the porcine epidemic diarrhea virus in a sample comprising one or more of porcine transmissible gastroenteritis virus, porcine acute diarrhea syndrome coronavirus, porcine delta coronavirus, porcine rotavirus, porcine respiratory coronavirus disease, and porcine sendai valley virus.
7. A method for detecting porcine epidemic diarrhea virus comprises the following steps:
1) extracting nucleic acid from a sample to be detected;
2) performing RT-PCR amplification by using the nucleic acid obtained in the step 1) as an amplification template and using the primer of claim 1 to obtain an amplification product;
3) and carrying out agarose gel electrophoresis on the amplification product to obtain a detection result.
8. The use of the primer for detecting porcine epidemic diarrhea virus according to claim 1, the kit for detecting porcine epidemic diarrhea virus according to claim 2, or the method for detecting porcine epidemic diarrhea virus according to claim 7 for detecting porcine epidemic diarrhea virus.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112760418A (en) * | 2021-01-30 | 2021-05-07 | 福建傲农生物科技集团股份有限公司 | Primer and kit for porcine epidemic diarrhea virus and application of primer and kit |
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| CN108060269A (en) * | 2018-01-19 | 2018-05-22 | 东北农业大学 | DPO primer sets and its application for the detection of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus |
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| CN107686864A (en) * | 2017-09-28 | 2018-02-13 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus TaqMan fluorescent quantitation RT PCR kits and its detection method |
| CN108060269A (en) * | 2018-01-19 | 2018-05-22 | 东北农业大学 | DPO primer sets and its application for the detection of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and porcine rotavirus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760418A (en) * | 2021-01-30 | 2021-05-07 | 福建傲农生物科技集团股份有限公司 | Primer and kit for porcine epidemic diarrhea virus and application of primer and kit |
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Application publication date: 20200724 |