CN111471802A - Porcine delta coronavirus rapid detection primer, kit and application thereof - Google Patents
Porcine delta coronavirus rapid detection primer, kit and application thereof Download PDFInfo
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- CN111471802A CN111471802A CN202010471895.2A CN202010471895A CN111471802A CN 111471802 A CN111471802 A CN 111471802A CN 202010471895 A CN202010471895 A CN 202010471895A CN 111471802 A CN111471802 A CN 111471802A
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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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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Abstract
The invention provides a primer and a kit for rapidly detecting porcine delta coronavirus and application thereof. The invention designs DPO primers according to highly conserved specific sequences of porcine delta coronavirus (PDCoV) by introducing a double Priming Oligonucleotide primer (DPO) design technology, and establishes a detection method aiming at the PDCoV and a corresponding detection kit through reaction system optimization. The kit has the characteristics of rapidness, convenience, strong specificity, high sensitivity and good reliability, can realize accurate detection on PDCoV and assist in prevention, control, diagnosis and treatment of PDCoV, 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 primer and a kit for rapidly detecting porcine delta coronavirus, and application thereof.
Background
Porcine delta coronavirus (PDCoV) is discovered in 2012 through epidemiological investigation, and then PDCoV is isolated and identified on LL C-PK cells and is a novel porcine coronavirus which can cause acute enteritis of newborn piglets.
PDCoV belongs to the genus coronaviridae (Coronavirinae), a enveloped single-stranded positive-strand RNA virus, which is the smallest Coronavirus known today, has a genome length of about 25.4kb, and consists of a 5 'untranslated region (UTR), 6 open reading frames (ORF1a, ORF1b, and ORFs 2 to 5), and a 3' UTR. The virus and other porcine coronavirus can cause symptoms of vomiting, diarrhea and the like of pigs clinically, and are difficult to distinguish from clinical characteristics and pathogenic forms.
At present, the diagnosis methods of domestic laboratories comprise pathological 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, have low sensitivity and are easy to generate false positive. In recent years, the focus of PDCoV detection research is mainly a PCR method which is strong in practicability, high-efficiency, rapid, accurate and widely applied, but the conventional PCR primer has a complex design process, needs to repeatedly optimize primer parameters, has poor specificity, and sometimes avoids the occurrence of non-specific amplification even if reaction conditions are optimized.
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, the complex operation of repeated optimization reaction in the traditional method is avoided, and the complex steps of conventional primer design are greatly simplified.
In view of the above, the present invention is expected to establish a PCR method for detecting PDCoV based on a DPO technique by introducing a double priming oligonucleotide primer (DPO) design technique to improve the sensitivity and specificity of detection and achieve accurate detection of PDCoV.
Disclosure of Invention
The invention provides a primer and a kit for rapidly detecting porcine delta coronavirus and application thereof, wherein a double-start oligonucleotide primer (DPO) design technology is introduced to compare gene sequences of PDCoV, a high-specificity sequence of the PDCoV is selected to design a DPO primer, and a high-sensitivity high-specificity detection method for the PDCoV is established to realize accurate detection of the PDCoV and help prevention, control and diagnosis of the PDCoV.
To this end, the first aspect of the present invention provides a primer for rapid detection of porcine delta coronavirus, which comprises:
an upstream primer: 5'-TCAACGCTAGAGGAAGACCTCAGIIIIITGGAAGTG-3' (SEQ ID NO.1),
a downstream primer: 5'-CATGATGCGAGGATCAGCCATAIIIIICTTCTCAG-3' (SEQ ID NO.2),
wherein I represents inosine.
The second aspect of the invention provides a rapid detection kit for porcine delta coronavirus, which comprises an upstream primer and a downstream primer shown in SEQ ID No.1 and SEQ ID No.2, and further comprises a positive quality control substance and a negative quality control substance.
In one embodiment of the invention, the final reaction concentration of the upstream primer and the downstream primer in a reaction system of the kit for rapidly detecting the porcine delta coronavirus is 0.05-0.6 mu M.
In a preferred embodiment of the invention, the final concentration of the upstream and downstream primer in the reaction system of the kit for rapidly detecting the porcine delta coronavirus is 0.1-0.6 mu M.
In an embodiment of the invention, the reaction conditions of the kit for rapidly detecting porcine delta coronavirus include:
in a preferred embodiment of the present invention, the reaction conditions of the rapid detection kit for porcine delta coronavirus include:
in an embodiment of the invention, the detection sensitivity of the rapid detection kit for porcine delta coronavirus can reach 7.47 × 103copies/μL。
In an embodiment of the present invention, the rapid detection kit for porcine delta coronavirus can specifically identify porcine delta coronavirus in a sample, wherein the sample comprises one or more of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine acute diarrhea syndrome coronavirus, porcine rotavirus, porcine respiratory coronavirus disease and porcine semaglaia valley virus.
The third aspect of the invention provides a rapid detection method for porcine delta coronavirus, which comprises the following steps:
1) extracting nucleic acid from a sample to be detected;
2) performing reverse transcription reaction by using the nucleic acid obtained in the step 1) as a template to obtain cDNA;
3) carrying out PCR amplification by using the cDNA obtained in the step 2) as an amplification template and using the primer of the first aspect of the invention to obtain an amplification product;
4) and carrying out agarose gel electrophoresis on the amplification product to obtain a detection result.
In one embodiment of the present invention, the reaction conditions of the reverse transcription reaction in step 2) are as follows:
in one embodiment of the present invention, the agarose gel of step 4) is a 1% agarose gel.
In a fourth aspect, the present invention provides a primer for rapidly detecting porcine delta coronavirus according to the first aspect of the present invention, a kit for rapidly detecting porcine delta coronavirus according to the second aspect of the present invention, or a method for rapidly detecting porcine delta coronavirus according to the third aspect of the present invention, for use in detecting porcine delta coronavirus.
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 delta coronavirus, so that the kit is suitable for monitoring and early warning of the trace latent infection period of the porcine delta coronavirus, 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 rapid detection method for porcine delta coronavirus provided in the embodiment of the present invention, wherein M is DNAMarker1000, and 1 to 6 are respectively: 42 ℃, 46 ℃, 50 ℃, 54 ℃, 58 ℃ and 62 ℃;
fig. 2 is a primer concentration optimization result of the rapid detection method for porcine delta coronavirus provided in the embodiment of the present invention, wherein M is DNAMarker1000, and 1 to 6 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 specificity test result of the rapid detection method for porcine delta coronavirus provided in the embodiment of the present invention, wherein M is DNAMarker1000, 1 is a positive control, and 2 to 7 are respectively: porcine transmissible gastroenteritis virus (TGEV), Porcine Epidemic Diarrhea Virus (PEDV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine rotavirus (PoRV), porcine respiratory coronavirus disease (PRCV) and porcine intrasertoli valley virus (SVV) samples, 8 is a negative control;
FIG. 4 shows the sensitivity test results of the rapid detection method for porcine delta coronavirus provided in the embodiment of the present invention, wherein M is DNAmarker1000, and 1-8 are respectively 10-fold samples diluted in gradient, and the dilution levels are as follows: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8；
Fig. 5 is a detection result of the rapid detection method for porcine delta coronavirus on a clinical sample, wherein M is DNAMarker1000, 1 is a negative control, 2 is a positive control, and 3-22 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 PCR Rapid detection method
1. Designing a primer:
based on the gene sequences registered in GenBank, the 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 PDCoV-DPO-F:
5'-TCAACGCTAGAGGAAGACCTCAGIIIIITGGAAGTG-3'(SEQ ID NO.1)，
a downstream primer PDCoV-DPO-R:
5'-CATGATGCGAGGATCAGCCATAIIIIICTTCTCAG-3'(SEQIDNO.2)，
wherein I represents inosine.
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. RNA reverse transcription:
and (3) carrying out reverse transcription on the RNA extracted in the step (2) as a template to prepare cDNA, wherein the steps and reagents are as follows:
4. and (3) PCR reaction:
PCR amplification was performed using the cDNA prepared in step 3 as a template and 2 × AccurateTaq premix (Cat. AG11010) from Aikery, Hunan as follows:
the reaction conditions were as follows:
5. and (3) judging the detection result, namely mixing the PCR product 5 mu L with loading Buffer 1 mu L, adding the mixture into an agarose gel electrophoresis spotting hole, adding 5 mu L D L1000 DNA Marker beside the sample hole as a control, carrying out electrophoresis by using 100V-130V voltage 50mA, observing the result under ultraviolet light after 15min-30min, identifying the amplification product by 1% agarose gel electrophoresis, and if a single-purpose strip with the size of 263bp can be amplified, judging that the sample is positive to the porcine delta coronavirus.
6. Gene cloning, construction of plasmid containing target gene
Extracting RNA of the porcine delta coronavirus, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification on a target gene, recovering and purifying a PCR product of the amplified target gene, connecting the purified PCR product with a PMD-19T vector overnight, and then transforming escherichia coli DH5 α.
7. 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.
8. 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 primer concentration optimization results are shown in fig. 2, and the effective concentrations of the DPO primers are as follows: 0.05-0.6 mu M, and when the concentration of the DPO primer is 0.1-0.6 mu M, the PCR amplification effect is better.
9. Specificity of porcine delta coronavirus rapid detection method
Detecting according to the reverse transcription and PCR reaction in the steps 3 and 4, respectively replacing sample template RNA with TGEV, PEDV, SADS-CoV, PoRV, PRCV and SVV samples, carrying out PCR detection by taking a porcine delta coronavirus sample which is detected to be positive in the laboratory and identified by sequencing as a positive control, identifying a PCR product by electrophoresis of 1% agarose gel, and displaying an electrophoresis result as shown in figure 3, wherein the result shows that a single band appears only at 263bp of the positive control lane, which indicates that the method has good specificity.
10. Sensitivity of porcine delta coronavirus rapid detection method
Detection was performed by reverse transcription and PCR reaction as described in step 3 and step 4, using RNA from the positive sample in step 9 (7.47 × 10)9copies/μ L) press 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8Performing 10-fold gradient dilution, performing PCR amplification with deionized water as negative control, identifying amplification 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 10-6In order to be the lowest detection limit, the porcine delta coronavirus rapid detection method provided by the invention can detect 7.47 × 10 at the lowest3copies/μL。
11. Clinical sample testing
Detecting according to the reverse transcription and PCR reaction in the steps 3 and 4, detecting 22 clinical samples suspected of generating the porcine delta coronavirus by taking the positive control RNA in the step 9 as a positive control and taking deionized water as a negative control, extracting the template RNA and performing PCR reaction in the steps 2 and 3, identifying the PCR product 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 6 samples were PDCoV positive.
In conclusion, the kit for rapidly detecting the porcine delta coronavirus has high detection sensitivity and specificity, has low requirements on equipment and personnel skills, is suitable for monitoring and early warning of a trace recessive infection period of the porcine delta coronavirus, 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 delta coronavirus, can accurately identify and diagnose the cause of the porcine infection, is beneficial to implementing 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
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Claims (8)
1. A primer for rapidly detecting porcine delta coronavirus is characterized by comprising the following components:
an upstream primer: 5'-TCAACGCTAGAGGAAGACCTCAGIIIIITGGAAGTG-3' (SEQ ID NO.1),
a downstream primer: 5'-CATGATGCGAGGATCAGCCATAIIIIICTTCTCAG-3' (SEQ ID NO.2),
wherein I represents inosine.
2. A rapid detection kit for porcine delta coronavirus 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 product and a negative quality control product.
3. The kit for rapidly detecting porcine deltacoronavirus according to claim 2, wherein the final reaction concentration of the upstream primer and the downstream primer in the reaction system of the kit is 0.05-0.6 μ M.
4. The rapid porcine deltacoronavirus detection kit of claim 2, wherein the kit reaction conditions comprise:
5. the kit for rapidly detecting porcine deltacoronavirus according to claim 2, wherein the detection sensitivity of the kit is up to 7.47 × 103copies/μL。
6. The kit for rapidly detecting porcine delta coronavirus according to claim 2, wherein the kit is capable of specifically identifying porcine delta coronavirus in a sample, wherein the sample comprises one or more of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine acute diarrhea syndrome coronavirus, porcine rotavirus, porcine respiratory coronavirus disease, and porcine semaglaia valley virus.
7. A method for rapidly detecting porcine delta coronavirus comprises the following steps:
1) extracting nucleic acid from a sample to be detected;
2) performing reverse transcription reaction by using the nucleic acid obtained in the step 1) as a template to obtain cDNA;
3) carrying out PCR amplification by using the cDNA obtained in the step 2) as an amplification template and using the primer of the first aspect of the invention to obtain an amplification product;
4) and carrying out agarose gel electrophoresis on the amplification product to obtain a detection result.
8. The use of the primer for rapid detection of porcine delta coronavirus according to claim 1, the kit for rapid detection of porcine delta coronavirus according to claim 2, or the method for rapid detection of porcine delta coronavirus according to claim 7 for detecting porcine delta coronavirus.
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| CN113046487A (en) * | 2021-04-30 | 2021-06-29 | 派生特（福州）生物科技有限公司 | Primer pair and kit for detecting porcine delta coronavirus |
| CN113151605A (en) * | 2021-04-30 | 2021-07-23 | 派生特（福州）生物科技有限公司 | Primer group and kit for nested RT-PCR (reverse transcription-polymerase chain reaction) detection of porcine delta coronavirus |
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| CN113046487A (en) * | 2021-04-30 | 2021-06-29 | 派生特（福州）生物科技有限公司 | Primer pair and kit for detecting porcine delta coronavirus |
| CN113151605A (en) * | 2021-04-30 | 2021-07-23 | 派生特（福州）生物科技有限公司 | Primer group and kit for nested RT-PCR (reverse transcription-polymerase chain reaction) detection of porcine delta coronavirus |
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