CN104818344A - General nucleic acid detecting method for coronaviruses - Google Patents
General nucleic acid detecting method for coronaviruses Download PDFInfo
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- CN104818344A CN104818344A CN201510250986.2A CN201510250986A CN104818344A CN 104818344 A CN104818344 A CN 104818344A CN 201510250986 A CN201510250986 A CN 201510250986A CN 104818344 A CN104818344 A CN 104818344A
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- 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
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- C—CHEMISTRY; METALLURGY
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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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Abstract
The invention belongs to the technical field of biology and determines a general nucleic acid detecting method for coronaviruses such as A coronavirus, B coronavirus, C coronavirus and D coronavirus. The general nucleic acid detecting method has the three technical key points as follows: (1) a primer sequence for nucleic acid detection is determined; (2) detection reaction kinds are determined; and (3) a detection reaction system and a reaction condition are determined. The general nucleic acid detecting method can be used for scientific research of coronavirus and clinical diagnosis and detection of animals and people.
Description
Technical field
The invention belongs to biological technical field; Particularly, the present invention establishes the universal test method of the important virus of a group (i.e. coronavirus), for first, second, third, the detection of coronavirus that belongs to of fourth etc. four, biological study, medical diagnosis and Disease Diagnosis of Veterinary have larger use value.
Background technology
Coronavirus is the important cause of disease of the animals such as people, fowl, dog, pig, can be divided into first coronavirus genus (
alphacoronavirus), second coronavirus genus (
betacoronavirus), the third coronavirus genus (
gammacoronavirus), fourth coronavirus genus (
deltacoronavirus) four belong to, each genus has again several coronavirus.
Detecting viral nucleic acid is detect one of viral important method.At present, be all only belong to or the coronavirus of a kind for one, not for the general nucleic acid detection method of the coronavirus of all genus to the various nucleic acid detection methods of coronavirus both at home and abroad.
Summary of the invention
The present invention is directed to the deficiency of above-mentioned existing coronavirus detection technique, carried out further investigation and test, establish first, second, third, four general nucleic acid detection methods of coronavirus belonged to such as fourth.The method comprises following three aspects:
(1) primer sequence required for detection of nucleic acids is established, conservative region in the genes such as coronary virus 1b, design primer sequence, wherein upstream primer sequence contains 25 bases, sequence is SEQ ID NO.1 in tgggttgggaytaycctaart gtga(and sequence table, wherein y and r is degeneracy base, y represents the mixture of t and c, r represents the mixture of g and a), downstream upstream primer sequence contains 23 bases, sequence is SEQ ID NO.2 in tgytgtgarcaraaytcatgwgg(and sequence table, wherein y, r and w is degeneracy base, y represents the mixture of t and c, r represents the mixture of g and a, w represents the mixture of a or t),
(2) detection reaction kind is established, multiple detection reaction can be adopted, comprise common reverse transcriptase chain reaction (usually, this kind of reaction product nucleic acid electrophoresis detects), fluorescence reverse transcriptase chain reaction (usually, this kind of reaction product fluorescent substance detects) or rely on amplification of nucleic acid sequences reaction (usually, this kind of reaction product electricity consumption chemiluminescent substance detects).
(3) detection reaction system and reaction conditions is established, for common reverse transcriptase chain reaction, pure water 7.5 microlitre is added successively in the pipe of polymerase chain reaction, 2 times of single stage method reverse transcriptase chain reaction damping fluid 12.5 microlitres are (containing dATP, dGTP, dTTP, four kinds of Nucleotide such as dCTP), mixture 1 microlitre of the reversed transcriptive enzyme required for single stage method reverse transcriptase chain reaction and nucleic acid polymerase, upstream primer 1 microlitre (concentration is that 10 micromoles often rise) synthesized by the first step, downstream primer 1 microlitre (concentration is that 10 micromoles often rise) synthesized by the first step, need coronavirus nucleic acid 2 microlitre (from clinical sample or other samples with nucleic acid extraction kit extract) detected, then by airtight for reverse transcriptase chain reaction system, be placed on Polymerized human serum albumin device (domestic be often called PCR instrument) and carry out reverse transcriptase chain reaction, reaction conditions be 50 degrees Celsius 30 minutes, then 94 degrees Celsius 2 minutes, carry out 40 circulations again, each circulation be 94 degrees Celsius 30 seconds, 55 degrees Celsius 30 seconds, 72 degrees Celsius 30 seconds, after loop ends 72 degrees Celsius extend 5 to 10 minutes, after reaction terminates, add in the reaction product containing coloured nucleic acid electrophoresis damping fluid, carry out agarose nucleic acid gel electrophoresis, be about the nucleic acid electrophoresis band of 520 to 570 base pairs if there is size, be then judged as the positive, otherwise be judged as feminine gender.
Embodiment
Below by embodiment, technical scheme of the present invention is described, but protection scope of the present invention is not limited to this embodiment.
The present embodiment reverse transcriptase chain reaction, carries out general detection of nucleic acids to coronavirus, comprises the steps:
The first step (synthetic primer): the nucleotide sequence (i.e. SEQ ID NO.1 and SEQ ID NO.2 in sequence table) of specifying according to the present invention, the upstream primer required for synthetic reverse transcriptase chain reaction and downstream primer;
Second step (configuration reverse transcriptase chain reaction system): add pure water 7.5 microlitre in the pipe of polymerase chain reaction successively, 2 times of single stage method reverse transcriptase chain reaction damping fluid 12.5 microlitres are (containing dATP, dGTP, dTTP, four kinds of Nucleotide such as dCTP), mixture 1 microlitre of the reversed transcriptive enzyme required for single stage method reverse transcriptase chain reaction and nucleic acid polymerase, upstream primer 1 microlitre (concentration is that 10 micromoles often rise) synthesized by the first step, downstream primer 1 microlitre (concentration is that 10 micromoles often rise) synthesized by the first step, need coronavirus nucleic acid 2 microlitre (from clinical sample or other samples with nucleic acid extraction kit extract) detected,
3rd step (carrying out reverse transcriptase chain reaction): after the reverse transcriptase chain reaction system configured by second step is airtight, be placed on Polymerized human serum albumin device (domestic be often called PCR instrument) and carry out reverse transcriptase chain reaction, reaction conditions be 50 degrees Celsius 30 minutes, then 94 degrees Celsius 2 minutes, carry out 40 circulations again, each circulation be 94 degrees Celsius 30 seconds, 55 degrees Celsius 30 seconds, 72 degrees Celsius 30 seconds, after loop ends 72 degrees Celsius extend 5 to 10 minutes;
4th step (detection of reverse transcriptase chain reaction result): add in the reaction product of the 3rd step containing coloured nucleic acid electrophoresis damping fluid, carry out agarose nucleic acid gel electrophoresis, the nucleic acid electrophoresis band of 520 to 570 base pairs is about if there is size, then be judged as the positive, otherwise be judged as feminine gender.
Result of practical application: to 1 part of human corona virus's standard positive clinical sample, to 2 parts of pig coronavirus standard positive clinical samples, 401 parts of cockscomb shape Virus Standard positive clinical samples, 82 parts of duck coronavirus standard positive clinical samples, 1 part of goose coronavirus standard positive clinical sample, 64 parts of pigeon coronavirus standard positive clinical samples, and 4592 parts of standard female samples from fowl and people, detect with the general nucleic acid detection technique of above-mentioned coronavirus, the sensitivity that result shows this technology is 99.64%, and specificity is 99.73%.
<110> China Animal Health and Epidemiology Center
The general nucleic acid detection method of <120> coronavirus
<160> 2
<210> 1
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> designs according to nucleotide sequence conservative in the genome of various coronavirus, as the upstream primer required for coronavirus detection of nucleic acids, wherein y and r is degeneracy base, and y represents the mixture of t and c, and r represents the mixture of g and a.
<400> 1
tgggttggga ytaycctaar tgtga 25
<210> 2
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> designs according to nucleotide sequence conservative in the genome of various coronavirus, as the downstream primer required for coronavirus detection of nucleic acids, wherein y, r and w are degeneracy bases, y represents the mixture of t and c, r represents the mixture of g and a, and w represents the mixture of a or t;
<400> 2
tgytgtgarc araaytcatg wgg 23
Claims (6)
1. the nucleic acid detection technique that coronavirus is general, common reverse transcriptase chain reaction, fluorescence reverse transcriptase chain reaction can be adopted, rely on the detection reaction such as amplification of nucleic acid sequences reaction, it is characterized in that detecting first, second, third, four coronavirus belonged to such as fourth.
2. the nucleic acid detection technique that coronavirus is general, its feature have two: one be can detect first, second, third, the coronavirus that belong to of fourth etc. four; Two is adopt common reverse transcriptase chain reaction or fluorescence reverse transcriptase chain reaction.
3. the nucleic acid detection technique that coronavirus is general, its feature have three: one be can detect first, second, third, the coronavirus that belong to of fourth etc. four; Two is adopt the region that for coronary virus 1b gene is guarded and the pair of primers designed; Three is adopt common reverse transcriptase chain reaction or fluorescence reverse transcriptase chain reaction.
4. the nucleic acid detection technique that coronavirus is general, reverse transcriptase chain reaction, fluorescence reverse transcriptase chain reaction can be adopted, rely on the detection reaction such as amplification of nucleic acid sequences reaction, its feature have two: one be can detect first, second, third, four coronavirus belonged to such as fourth; Two are sequences of a primer that detection reaction adopts is tgggttgggaytaycctaartgtga, or have sequence that the base sequence of more than 15 or 15 is identical (in this section of word with tgggttgggaytaycctaartgtga sequence, primer sequence is 5 slash ends to 3 and skims end, a, t, g, c represent corresponding base, y and r is degeneracy base, y represents the mixture of t and c, and r represents the mixture of g and a).
5. the nucleic acid detection technique that coronavirus is general, reverse transcriptase chain reaction, fluorescence reverse transcriptase chain reaction can be adopted, rely on the detection reaction such as amplification of nucleic acid sequences reaction, its feature have two: one be can detect first, second, third, four coronavirus belonged to such as fourth; Two are sequences of a primer that detection reaction adopts is tgytgtgarcaraaytcatgwgg, or have sequence that the base sequence of more than 15 or 15 is identical (in this section of word with tgytgtgarcaraaytcatgwgg sequence, primer sequence is 5 slash ends to 3 and skims end, a, t, g, c represent corresponding base, y, r and w are degeneracy bases, y represents the mixture of t and c, and r represents the mixture of g and a, and w represents the mixture of a or t).
6. the nucleic acid detection technique that coronavirus is general, its feature has three: one to be adopt common reverse transcriptase chain reaction or fluorescence reverse transcriptase chain reaction; Two is two primers that detection reaction adopts, its sequence is respectively ccagttggwggtaatgaaaa and gttgacagcatgttraacatkcccat, or have sequence that the base sequence of more than 15 or 15 is identical (in this section of word with these two sequences, primer sequence is 5 slash ends to 3 and skims end, a, t, g, c represent corresponding base, w, r and k represent degeneracy base, w represents the mixture of base a and base t, r represents the mixture of base g and base a, and k represents the mixture of base g and base t).
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1239548A (en) * | 1997-08-04 | 1999-12-22 | 东燃株式会社 | Methods for detecting or assaying virus |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1239548A (en) * | 1997-08-04 | 1999-12-22 | 东燃株式会社 | Methods for detecting or assaying virus |
Non-Patent Citations (2)
| Title |
|---|
| 邹丽容等: "用 RT - PCR法鉴定人冠状病毒SARS、229E和OC43", 《中国卫生检验杂志》 * |
| 魏桂芳等: "从粪便样品中检测动物冠状病毒的分子技术研究", 《中国微生态学杂志》 * |
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