CN112505324A - Novel coronavirus detection method - Google Patents

Abstract

The invention discloses a novel coronavirus detection method, which comprises the following steps: the method comprises the following steps: obtaining a nucleic acid sequence of a target gene; step two: performing PCR amplification on the nucleic acid sequence of the synthetic target gene by adopting a PCR primer containing a tag sequence; step three: putting the PCR product into an in vitro transcription and translation system to obtain a virus protein with a tag; step four: anchoring the tagged viral protein to the surface of the affinity medium to obtain a viral protein bound to the affinity medium; step five: antibodies in the blood of the patient are detected using viral proteins bound to the affinity medium. By adopting the arrangement, the nucleic acid detection and the protein detection are connected in series through the in vitro transcription and translation system, so that the method has the characteristic of high accuracy of nucleic acid detection, can obtain information such as infection time of a patient and the like, and has high detection speed and high efficiency.

Description

Novel coronavirus detection method

Technical Field

The invention relates to the technical field of novel coronary pneumonia diagnosis, in particular to a novel coronavirus detection method.

Background

The current popular virus detection method, including the detection method of the new coronavirus (Covid-19), generally adopts nucleic acid detection and protein detection. The nucleic acid detection is that the virus gene in the blood of target patient is amplified through DNA polymerase chain reaction, and whether the detected target is infected with the virus is judged through detecting the existence of the amplified product. Protein detection is carried out by immunological means, generally by detecting an antibody or an antigen in blood of a target to be detected by an antibody-antigen interaction in combination with a fluorescence or color development method. The two detection methods have the advantages and disadvantages, such as high accuracy of nucleic acid detection, long time consumption, incapability of judging the time of infecting the target patient with viruses, short time consumption of the protein detection method, low accuracy of protein detection and frequent occurrence of false positive phenomenon, and the infection time of the target patient can be judged by detecting IgG and IgM.

Disclosure of Invention

The invention aims to provide a novel coronavirus detection method which has the advantages of accurate detection and high detection speed.

The technical purpose of the invention is realized by the following technical scheme:

a novel coronavirus detection method comprises the following steps:

the method comprises the following steps: obtaining a nucleic acid sequence of a target gene;

step two: performing PCR amplification on the nucleic acid sequence of the synthetic target gene by adopting a PCR primer containing a tag sequence;

step three: putting the PCR product into an in vitro transcription and translation system to obtain a virus protein with a tag;

step four: anchoring the tagged viral protein to the surface of the affinity medium to obtain a viral protein bound to the affinity medium;

step five: antibodies in the blood of the patient are detected using viral proteins bound to the affinity medium.

By adopting the technical scheme, virus particles are obtained by extracting peripheral blood of a known patient, and virus deoxyribonucleic acid is obtained by extraction, or the virus deoxyribonucleic acid is obtained by a virus library; then obtaining virus protein by PCR amplification, anchoring the virus protein on the affinity medium, and detecting the antibody of the patient; the in vitro transcription and translation system has the advantages of high synthesis speed, high synthesis quality, a living cell in vivo expression system and a cell-free protein synthesis system, no membrane barrier, high controllability, easy quantitative measurement of a reporter and convenient subsequent use.

Further setting: and in the fifth step, the detection method adopts ELISA detection.

By adopting the technical scheme, the ELISA method is based on the immunological reaction of the antigen and the antibody, so that the ELISA method has specificity; because the enzyme-labeled antigen or antibody is the conjugate of enzyme molecules and antigen or antibody molecules, it can catalyze the reaction of substrate molecules to produce amplification action, and because of the amplification action said method possesses high sensitivity, and the ELISA possesses the characteristics of simple, quick, stable and easy to implement automatic operation, so that it is applicable to large-scale specimen detection.

Further setting: the PCR primer in the second step also comprises a transcription promoter sequence, a ribosome binding site and a transcription promoter sequence.

Further setting: the transcription promoter sequence includes, but is not limited to, the T7 promoter sequence.

Further setting: and in the second step, the tag sequence comprises one or more of histidine, Gramicidin, SUMO, FLAG, Biotin, His6 and ubiquitin.

Further setting: the ambient temperature was set at 15-40 degrees celsius throughout the steps.

Further setting: and step four, adding one or more of detergents, phospholipids, nanodiscs and molecular chaperones.

Further setting: and step two, adding T7 RNA polymerase, ribosome and tRNA into the PCR reaction system.

In conclusion, the invention has the following beneficial effects:

the invention connects the nucleic acid detection and the protein detection in series through an in vitro transcription and translation system, has the characteristic of high accuracy of the nucleic acid detection, and can obtain the information of the infection time of a patient and the like. The method can also be used as a preparation method of virus recombinant protein, can obtain virus antigen within 24 hours, and shortens the preparation time by tens of times compared with the preparation time of the traditional recombinant protein expression method.

Drawings

FIG. 1 is a graph showing the results of PCR amplification using primers carrying His6 tag gene sequences;

FIG. 2 is a diagram showing the results of western blotting detection.

Detailed Description

Example 1:

a novel coronavirus detection method comprises the following steps:

the method comprises the following steps: the virus particles are obtained by extracting the peripheral blood of a known patient, and the nucleic acid sequence of the target gene is obtained by extraction.

Step two: and performing PCR amplification on the nucleic acid sequence of the synthetic target gene by using a PCR primer containing a tag sequence, wherein T7 RNA polymerase, ribosome and tRNA are added into a PCR reaction system during amplification.

The primer comprises a T7 promoter sequence, a ribosome binding site and a transcription promoter sequence, wherein the tag sequence is a histidine tag sequence, and the T7 promoter sequence is TAATACGACTCACTATAG.

The sequences of the PCR primers were:

5 '-TAATACGACTCACTAXXxxxxxxxxxxTTGTTAACTTTAAGAAGGAGAXX xxxxxxxxATGCACCATCACCATCACCATCACCAT-viral nucleic acid sequence-3';

5 '-AAAAAAAAAAAAAATTA-viral nucleic acid sequence-3'.

Step three: placing the PCR product in an in vitro transcription and translation system, wherein the in vitro transcription and translation system is added with an additive detergent, phospholipid and a nanodisk to obtain a tagged viral protein;

step four: anchoring the tagged viral protein to the surface of the affinity medium to obtain a viral protein bound to the affinity medium;

step five: the antibodies in the blood of the patient are detected by ELISA using viral proteins bound to an affinity medium.

The steps are carried out in an environment of 25 ℃.

Example 2: a novel coronavirus detection method comprises the following steps:

the method comprises the following steps: the nucleic acid sequence of the target gene is obtained from the virus library.

Step two: and performing PCR amplification on the nucleic acid sequence of the synthetic target gene by using a PCR primer containing a tag sequence, wherein T7 RNA polymerase, ribosome and tRNA are added into a PCR reaction system during amplification.

The primer comprises a T7 promoter sequence, a ribosome binding site and a transcription promoter sequence, wherein the tag sequence is a FLAG tag sequence, and the T7 promoter sequence is TAATACGACTCACTATAG.

The sequences of the PCR primers were:

5 '-TAATACGACTCACTATGxxxxxxxxxxxxTTGTTAACTTTAAGAAGGAGAXX xxxxxxATGGATTACAAGGACGATGACAAG-viral nucleic acid sequence-3';

5 '-AAAAAAAAAAAAAATTA-viral nucleic acid sequence-3'.

Step three: placing the PCR product in an in vitro transcription and translation system, wherein the in vitro transcription and translation system is added with an additive detergent, a molecular chaperone and a nanodisk to obtain a tagged viral protein;

step four: anchoring the tagged viral protein to the surface of the affinity medium to obtain a viral protein bound to the affinity medium;

step five: the antibodies in the blood of the patient are detected by ELISA using viral proteins bound to an affinity medium.

The steps are carried out in an environment of 40 ℃.

The nucleic acid sequence of the target gene in example 1 and example 2 is one of the following viral nucleic acid sequences:

SEQ ID NO.1:

MADSNGTITV EELKKLLEQW NLVIGFLFLT WICLLQFAYA NRNRFLYIIK LIFLWLLWPV TLACFVLAAV YRINWITGGI AIAMACLVGL MWLSYFIASF RLFARTRSMW SFNPETNILL NVPLHGTILT RPLLESELVI GAVILRGHLR IAGHHLGRCD IKDLPKEITV ATSRTLSYYK LGASQRVAGD SGFAAYSRYR IGNYKLNTDH SSSSDNIALL VQ

SEQ ID NO.2:

MSDNGPQNQR NAPRITFGGP SDSTGSNQNG ERSGARSKQR RPQGLPNNTA SWFTALTQHG KEDLKFPRGQ GVPINTNSSP DDQIGYYRRA TRRIRGGDGK MKDLSPRWYF YYLGTGPEAG LPYGANKDGI IWVATEGALN TPKDHIGTRN PANNAAIVLQ LPQGTTLPKG FYAEGSRGGS QASSRSSSRS RNSSRNSTPG SSRGTSPARM AGNGGDAALA LLLLDRLNQL ESKMSGKGQQ QQGQTVTKKS AAEASKKPRQ KRTATKAYNV TQAFGRRGPE QTQGNFGDQE LIRQGTDYKH WPQIAQFAPS ASAFFGMSRI GMEVTPSGTW LTYTGAIKLD DKDPNFKDQV ILLNKHIDAY KTFPPTEPKK DKKKKADETQ ALPQRQKKQQ TVTLLPAADL DDFSKQLQQS MSSADSTQA

SEQ ID NO.3：

MYSFVSEETG TLIVNSVLLF LAFVVFLLVT LAILTALRLC AYCCNIVNVS LVKPSFYVYS RVKNLNSSRV PDLLV

SEQ ID NO.4：

MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PRRARSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQYI KWPWYIWLGF IAGLIAIVMV TIMLCCMTSC CSCLKGCCSC GSCCKFDEDD SEPVLKGVKL HYT

SEQ ID NO.5：

MKIILFLALI TLATCELYHY QECVRGTTVL LKEPCSSGTY EGNSPFHPLA DNKFALTCFS TQFAFACPDG VKHVYQLRAR SVSPKLFIRQ EEVQELYSPI FLIVAAIVFI TLCFTLKRKT E

SEQ ID NO.6：

MGYINVFAFP FTIYSLLLCR MNSRNYIAQV DVVNFNLT

SEQ ID NO.7：

MKFLVFLGII TTVAAFHQEC SLQSCTQHQP YVVDDPCPIH FYSKWYIRVG ARKSAPLIEL CVDEAGSKSP IQYIDIGNYT VSCSPFTINC QEPKLGSLVV RCSFYEDFLE YHDVRVVLDF I

example 3:

primers carrying the His6 tag gene sequence were used to perform PCR amplification of the following genes:

the results of PCR amplification are shown in FIG. 1, and it can be seen that the gene segments 7, 8, 9, 10, 11, 12 and 13 were successfully obtained.

The PCR product was used as a DNA template, added to an in vitro transcription and translation system, and the transcription and translation samples were subjected to western blotting detection (using His6 antibody) analysis, and the results are shown in FIG. 2, which shows that proteins Nos. 1, 2, 8, 9 and 12 were successfully obtained.

Anchoring the protein on a Ni-NTA enzyme label plate, then dripping novel coronavirus positive serum, carrying out ELISA detection by using anti-igG secondary antibody, and simultaneously carrying out a control example of the novel coronavirus negative serum, wherein the detection result shows that samples No. 1, 2, 8, 9 and 12 all obtain light absorption of 450nm which is higher than that of a negative control sample, wherein the sample No. 9 is obviously higher than that of the negative control sample, which indicates that the detection method is feasible and can be used for detecting the novel coronavirus.

450nm light absorption detection result of sample

The above-described embodiments do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the above-described embodiments should be included in the protection scope of the technical solution.

SEQUENCE LISTING

<110> Hangzhou Xuanxue Biotechnology Co., Ltd

<120> a novel coronavirus detection method

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<160> 9

<170> PatentIn version 3.3

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taatacgact cactattttg tttaacttta agaaggagaa tgcaccatca ccatcaccat 60

aaaaaaaaaa aaaatta 77

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Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu

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Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile

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Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile

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Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys

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Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile

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Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

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Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe

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Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile

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Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile

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Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp

145 150 155 160

Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu

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Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly

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Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr

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Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln

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Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

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Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

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Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

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Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

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Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

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Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

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Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

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Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

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Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

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Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

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Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

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Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

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Thr Gln Ala

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Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

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Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

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Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

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Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

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Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val

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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

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Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

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Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

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Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

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Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

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Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

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Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

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Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

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Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

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Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

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Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

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Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

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Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

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Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

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Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

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His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

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Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

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Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

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Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

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Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

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Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

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Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

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Met Lys Ile Ile Leu Phe Leu Ala Leu Ile Thr Leu Ala Thr Cys Glu

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Leu Tyr His Tyr Gln Glu Cys Val Arg Gly Thr Thr Val Leu Leu Lys

20 25 30

Glu Pro Cys Ser Ser Gly Thr Tyr Glu Gly Asn Ser Pro Phe His Pro

35 40 45

Leu Ala Asp Asn Lys Phe Ala Leu Thr Cys Phe Ser Thr Gln Phe Ala

50 55 60

Phe Ala Cys Pro Asp Gly Val Lys His Val Tyr Gln Leu Arg Ala Arg

65 70 75 80

Ser Val Ser Pro Lys Leu Phe Ile Arg Gln Glu Glu Val Gln Glu Leu

85 90 95

Tyr Ser Pro Ile Phe Leu Ile Val Ala Ala Ile Val Phe Ile Thr Leu

100 105 110

Cys Phe Thr Leu Lys Arg Lys Thr Glu

115 120

<210> 8

<211> 38

<212> PRT

<213> Natural

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Met Gly Tyr Ile Asn Val Phe Ala Phe Pro Phe Thr Ile Tyr Ser Leu

1 5 10 15

Leu Leu Cys Arg Met Asn Ser Arg Asn Tyr Ile Ala Gln Val Asp Val

20 25 30

Val Asn Phe Asn Leu Thr

35

<210> 9

<211> 121

<212> PRT

<213> Natural

<400> 9

Met Lys Phe Leu Val Phe Leu Gly Ile Ile Thr Thr Val Ala Ala Phe

1 5 10 15

His Gln Glu Cys Ser Leu Gln Ser Cys Thr Gln His Gln Pro Tyr Val

20 25 30

Val Asp Asp Pro Cys Pro Ile His Phe Tyr Ser Lys Trp Tyr Ile Arg

35 40 45

Val Gly Ala Arg Lys Ser Ala Pro Leu Ile Glu Leu Cys Val Asp Glu

50 55 60

Ala Gly Ser Lys Ser Pro Ile Gln Tyr Ile Asp Ile Gly Asn Tyr Thr

65 70 75 80

Val Ser Cys Ser Pro Phe Thr Ile Asn Cys Gln Glu Pro Lys Leu Gly

85 90 95

Ser Leu Val Val Arg Cys Ser Phe Tyr Glu Asp Phe Leu Glu Tyr His

100 105 110

Asp Val Arg Val Val Leu Asp Phe Ile

115 120

Claims (8)

1. A novel coronavirus detection method is characterized by comprising the following steps:

the method comprises the following steps: obtaining a nucleic acid sequence of a target gene;

step two: performing PCR amplification on the nucleic acid sequence of the synthetic target gene by adopting a PCR primer containing a tag sequence;

step three: putting the PCR product into an in vitro transcription and translation system to obtain a virus protein with a tag;

step four: anchoring the tagged viral protein to the surface of the affinity medium to obtain a viral protein bound to the affinity medium;

step five: antibodies in the blood of the patient are detected using viral proteins bound to the affinity medium.

2. The method of claim 1, wherein the detection method in step five is ELISA detection.

3. The method of claim 1, wherein the PCR primers in step two further comprise a transcription promoter sequence, a ribosome binding site and a transcription promoter sequence.

4. The method of claim 3, wherein the transcription promoter sequence includes but is not limited to T7 promoter sequence.

5. The method of claim 1, wherein the tag sequence in step two comprises one or more of histidine, graminidin, SUMO, FLAG, Biotin and ubiquitin.

6. The method of claim 1, wherein the ambient temperature is set to 15-40 degrees centigrade.

7. The method of claim 1, wherein one or more of detergents, phospholipids, nanodiscs and molecular chaperones are added to the step three.

8. The method of claim 1, wherein T7 RNA polymerase, ribosome, and tRNA are added to the PCR reaction system in the second step.

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