CN112063752A - Universal coronavirus PCR primer and application thereof - Google Patents
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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
- 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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- 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
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a universal coronavirus PCR primer and application thereof, wherein the primer comprises the following components in parts by weight: upstream primer P24F: 5'-AACTCAAGCCTTACCGCAGA-3', respectively; downstream primer P24R: 5'-ATAGCCCATCTGCCTTGTGT-3' are provided. The universal coronavirus PCR primer provided by the invention can be used for judging the result only by using a common PCR reagent and an electrophoresis method, and has the advantages of simple operation and low cost. Through PCR verification of various animal pharynx swabs, anus swabs, excrement and lymph node samples, the specificity is strong, the kit can be practically applied to identification of various animals carrying or infected with coronavirus, has the characteristics of high specificity, high sensitivity, low cost and simple operation, and can be applied to specificity detection of coronavirus in animal samples.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a universal coronavirus PCR primer and application thereof.
Background
Over 40 new infections have occurred worldwide since the 70's of the 20 th century, with over 80% of new infections originating from wild animals. Animal-derived epidemic diseases are increasingly paid more attention from countries in the world as an important public health problem for human beings. In recent years, with the progress of tracing research on infectious diseases, it is found that wild animal diseases are directly related to human health.
Coronaviruses (Coronavirus) are a group of viruses that cause diseases of digestive tracts and respiratory tracts in animals and humans, and they are a large family, and more than 30 kinds of them have been found, and are the largest viruses among RNA viruses, and the genome is about 30000 nucleotides in size. The diameter is 60-140 nm, and the virus is a single-stranded positive-sense RNA virus with an envelope. The subfamily coronaviruses are divided into 4 genera, i.e., α, β, γ and. Coronaviruses are common in wild animals, and about 29 kinds of alpha and beta coronaviruses are found at present and mainly infect human and veterinary animals. The currently known beta coronavirus includes SARS coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), Murine Hepatitis Virus (MHV), etc., and the new coronavirus (SARS-CoV-2) of this outbreak also belongs to the beta coronavirus. Gamma and coronavirus mainly infect birds, but it has also been found that gamma and coronavirus can infect beasts, for example gamma coronavirus can infect sea lions and coronavirus can infect pigs, causing disease death in domestic animals. It is important that coronaviruses are also capable of transmission across species, and once infectivity is enhanced, serious harm is done to animals as well as humans. In order to discover pathogens in a timely manner, cut off pathogen transmission, and avoid losses, there is an urgent need for rapid identification of coronaviruses, especially early identification. The traditional identification method mainly adopts means such as fluorescent quantitative primers and probes, ELASA antibody detection and the like, has strong technical performance and high cost, so that a coronavirus detection method which has low cost, low requirements on instruments and technicians and simple operation is urgently needed to quickly and accurately identify whether animals carry or infect coronaviruses, thereby playing a role in early warning for preventing the coronaviruses.
Disclosure of Invention
The first purpose of the invention is to provide a universal coronavirus PCR primer, which is as follows:
upstream primer P24F: 5'-AACTCAAGCCTTACCGCAGA-3', as shown in SEQ ID NO. 1;
downstream primer P24R: 5'-ATAGCCCATCTGCCTTGTGT-3', as shown in SEQ ID NO. 2.
The second purpose of the invention is to provide the application of the universal coronavirus PCR primer in the preparation of a kit for detecting coronavirus.
The third purpose of the invention is to provide a kit for detecting coronavirus, which comprises the universal coronavirus PCR primer.
It is a fourth object of the present invention to provide a method for detecting coronavirus, comprising the steps of: extracting virus RNA of a sample to be detected, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using the cDNA of the sample to be detected as a template and the universal coronavirus PCR primer P24F/P24R, and identifying whether the coronavirus exists.
The PCR reaction system is as follows: PCR Mix 12.5. mu.L, ddH2O10.5. mu.L, template 1. mu.L, upstream and downstream primers 10. mu.M each 0.5. mu.L, total 25. mu.L.
The PCR amplification conditions are as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃.
The sample to be detected comprises a throat swab, an anus swab, excrement or lymph nodes of an animal.
Compared with the prior art, the invention has the advantages that:
the existing detection method of coronavirus mainly utilizes fluorescent quantitative PCR primers and probes or ELASA antibody for detection, and has strong technical property and high cost. In order to rapidly, highly sensitively and inexpensively identify whether animals carry or are infected with coronavirus from a sample containing a small amount of viruses, the invention provides a universal coronavirus PCR primer, the result can be judged only by using a common PCR reagent and an electrophoresis method, the operation is simple, and the cost is low. Through PCR verification of various animal pharynx swabs, anus swabs, excrement and lymph node samples, the specificity is strong, the kit can be practically applied to identification of various animals carrying or infected with coronavirus, has the characteristics of high specificity, high sensitivity, low cost and simple operation, and can be applied to specificity detection of coronavirus in animal samples.
Drawings
FIG. 1 shows the result of specific detection of coronavirus universal primers. In the figure, M: marker; 1 is a fecal sample of healthy manis pentadactyla; 2 lymph node samples of dead manis pentadactyla; 3-4 is an anal swab sample of the horsetail bats; 5-6 are pharyngeal swab samples of the garter snake; 7 is a pharyngeal swab sample of a bamboo rat; negative control (ddH) 82O）。
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. A universal coronavirus PCR primer: upstream primer P24F: 5' -AACTCAAGCCTTACCGCAG
A-3', downstream primer P24R: 5'-ATAGCCCATCTGCCTTGTGT-3' are provided.
2. The detected sample is as follows: collecting throat swab, anal swab, excrement and the like of the living animal by using a sterile cotton swab aiming at the living animal; for the dead individual, visceral tissue was collected for its dissection. The samples identified in this example were feces of 1 healthy equine squama manitis (number 1), lymph nodes of 1 dead equine squama manitis (number 2), anal swabs of 2 individual horsehead bats (numbers 3 and 4), pharyngeal swabs of 2 gliding snakes (numbers 5 and 6), and pharyngeal swabs of 1 bamboo rat (number 7).
3. Obtaining cDNA: extracting RNA viruses from a sample by using a virus extraction Kit (QIAamp Viral RNA Mini Kit), and performing reverse transcription according to the instructions of a reverse transcription Kit (SuperScript III First-Strand Synthesis System) to synthesize single-stranded cDNA;
4. and (3) PCR amplification: using cDNA as a template, and using common high-efficiency PCR Mix to perform PCR amplification, wherein the PCR system is as follows: PCR Mix 12.5 μL，ddH210.5 mu L of O, 1 mu L of template, 0.5 mu L of each of upstream and downstream primers (10 mu M), and 25 mu L of total system; the amplification conditions were: 3min at 94 ℃; 35 cycles (94 ℃ 30s, 55 ℃ 30s, 72 ℃ 30 s); 5min at 72 ℃; the size of the band of interest for PCR amplification is about 160 nt. The PCR product was identified by 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1.
5. Sequencing and verifying: and (3) cutting, recovering and purifying the PCR product, sending the PCR product to a company for sequencing, and analyzing and identifying that the sequences are all beta-coronavirus. And (3) sequencing results: the sequencing result of the feces PCR product of 1 healthy manis pentadactyla is shown in SEQ ID NO. 3; the sequencing result of the lymph node PCR product of 1 dead manis pentadactyla is shown as SEQ ID NO.4, and the similarity of the virus sequence carried by the manis pentadactyla and the manis pentadactyla coronavirus is 100 percent through comparison and analysis of a NCBI database BLAST; the sequencing result of the anal swab PCR products of 2 horseshoe bats is shown in SEQ ID No.5-6, and through the comparison and analysis of NCBI database BLAST, the similarity of the virus sequence carried by the bat and the bat SARS coronavirus is the highest, and is 98.77%; the sequencing result of the pharyngeal swab PCR products of 2 mouse snakes is shown in SEQ ID NO.7-8, and the similarity of the virus sequence carried by the mouse snakes and the pangolin coronavirus is the highest and is 99.38 percent through comparison and analysis of NCBI database BLAST; the sequencing result of the pharyngeal swab PCR product of 1 bamboo rat is shown in SEQ ID NO.9, and through comparison and analysis of NCBI database BLAST, the similarity of the virus sequence carried by the bamboo rat and the pangolin coronavirus is the highest and is 99.38%.
The results of the PCR verification of various animal throat swabs, anal swabs, feces and lymph node samples show that the universal coronavirus PCR primer provided by the invention has strong specificity, can be practically applied to the identification of various animal-carried or infected coronaviruses, has the characteristics of high specificity, high sensitivity, low cost and simple operation, and can be applied to the specificity detection of coronaviruses in animal samples.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
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Claims (7)
1. A universal coronavirus PCR primer is characterized in that the primer is as follows:
upstream primer P24F: 5'-AACTCAAGCCTTACCGCAGA-3', respectively;
downstream primer P24R: 5'-ATAGCCCATCTGCCTTGTGT-3' are provided.
2. Use of the primer of claim 1 for the preparation of a kit for detecting coronaviruses.
3. A kit for detecting coronavirus, comprising the primer of claim 1.
4. A method for detecting coronaviruses, comprising the steps of: extracting virus RNA of a sample to be detected, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using the cDNA of the sample to be detected as a template and using the universal coronavirus PCR primer P24F/P24R as claimed in claim 1, and identifying whether the sample contains coronavirus.
5. The method for detecting coronavirus according to claim 4, wherein the PCR reaction system is: PCR Mix 12.5. mu.L, ddH2O10.5. mu.L, template 1. mu.L, upstream and downstream primers 10. mu.M each 0.5. mu.L, total 25. mu.L.
6. The method of claim 4, wherein the PCR amplification conditions are: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃.
7. The method according to claim 4, wherein the sample to be tested comprises a throat swab, an anal swab, feces or a lymph node of the animal.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113846186A (en) * | 2021-10-20 | 2021-12-28 | 云南农业大学 | Universal detection primer and detection method for animal beta coronavirus |
| CN117660702A (en) * | 2024-02-01 | 2024-03-08 | 广东省林业科学研究院 | Fluorescent quantitative PCR primer group and method for detecting Liquorice pangolin virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113846186A (en) * | 2021-10-20 | 2021-12-28 | 云南农业大学 | Universal detection primer and detection method for animal beta coronavirus |
| CN117660702A (en) * | 2024-02-01 | 2024-03-08 | 广东省林业科学研究院 | Fluorescent quantitative PCR primer group and method for detecting Liquorice pangolin virus |
| CN117660702B (en) * | 2024-02-01 | 2024-04-30 | 广东省林业科学研究院 | Fluorescent quantitative PCR primer group and method for detecting Liquorice pangolin virus |
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