CN103484564B - High-sensitivity method used for detecting and identifying human coronavirus - Google Patents

High-sensitivity method used for detecting and identifying human coronavirus Download PDF

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CN103484564B
CN103484564B CN201310309972.4A CN201310309972A CN103484564B CN 103484564 B CN103484564 B CN 103484564B CN 201310309972 A CN201310309972 A CN 201310309972A CN 103484564 B CN103484564 B CN 103484564B
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彭俊平
郭军华
金奇
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Institute of Pathogen Biology of CAMS
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Abstract

The invention provides a high-sensitivity method used for detecting and identifying 6 kinds of human coronavirus. The high-sensitivity method is based on exclusion of pollution caused by PCR amplification products, and is used for detecting and/or identifying human coronavirus by taking multiplex PCR-mass spectrometry as a platform. The 6 kinds of human coronavirus include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Detection parts are the specific sequence domains in the genomes of the 6 kinds of human coronavirus. The high-sensitivity method is capable of realizing multiple gene detection of the 6 kinds of human coronavirus in a reaction, and increasing detectable rate; possesses advantages of high sensitivity and specificity, convenient operation, speediness, high throughput and the like; and can be used in scientific researches, clinical and epidemiological investigation.

Description

A kind of method of high-sensitivity detection and identifier coronavirus
Technical field
The present invention relates to a kind of detection method of pathogenic micro-organism, particularly a kind of method of high-sensitivity detection and identifier coronavirus.
Background technology
Human corona virus (human coronavirus, HCoV) belongs to coronaviridae (Coronaviridae) coronavirus genus (Coronavirus), is strand justice RNA viruses.It is irregularly shaped that virus particle is, outsourcing panniculus, and there are three kinds of glycoprotein on film surface: spike glycoprotein (S, Spike Protein); Little envelope glycoprotein (E, Envelope Protein); Membrane glycoprotein (M, Membrane Protein).
HCoV is one of main pathogen of adult's common cold, children, can cause upper respiratory tract infection, generally seldom involves lower respiratory tract.The HCoV having found at present has 6 kinds: HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV.Wherein, the HCoV-229E and the HCoV-OC43 that find the earliest mainly cause upper respiratory tract infection, and symptom is lighter.In by the end of November, 2002, in China In Guangdong Province, occurred a kind of unknown cause to take closely that the air spittle and close contact propagates be main respiratory tract acute infectious disease, the domestic severe acute respiratory syndrome that is referred to as, i.e. SARS; The World Health Organization levies its called after serious acute respiratory system synthesis (Severe Acute Respiratory Syndrome, SARS), and rear confirmation is caused by SARS-CoV.SARS is newfound a kind of serious transmissible disease in this century, broadcasts and to more than 30 countries and regions, wherein take South East Asia as main epidemic-stricken area.Afterwards, HCoV-NL63, HCoV-HKU1 is found in succession, and wherein HCoV-NL63 can cause upper respiratory tract infection, and while infecting children's, can there is serious lower respiratory tract symptom in bronchiolitis and pneumonia; And HCoV-HKU1 infection shows as bronchiolitis and pneumonia more.In September, 2012, a kind of new coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV) be found, it can cause that mankind's serious acute respiratory infects (severe acute respiratory infection, SARI), end on June 18th, 2013, found 64 patients, wherein 38 people are dead.
Because HCoV can cause serious respiratory system disease, therefore its rapid detection is seemed to particularly important.Classical detection method comprises that virus culture and serology detect, but due to length consuming time, sensitivity is low, can not meet clinical demand.RT-PCR based on detection of nucleic acids and fluorescence quantifying PCR method become new main detection means, and conventional method, for single virus, because flux is low, causes testing amount to strengthen, and has had a strong impact on the further investigation of HCoV and human diseases relation.Therefore develop high-throughput multi detection method become in the urgent need to.As Chinese patent application CN102732638A takes 5 kinds of fluorescence, to HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and HCoV-HKU1 detect, and under this background, produce.But because this method need to be used fluorescence dye, actually operating is had relatively high expectations.And this method only adopts a probe to detect for every kind of HCoV.Because HCoV genome exists definitive variation, this method can cause to a certain degree undetected.The method at present 6 kinds of HCoV being detected in a reaction has no report.
Summary of the invention
For overcoming the defect of above-mentioned technology, the invention provides a kind of method that highly sensitive, high-throughput, employing multiprobe based on mass spectrum platform can detect 6 kinds of HCoV simultaneously.
6 kinds of HCoV that the present invention detects comprise: HCoV-229E, and HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV, detecting position is distinguished sequence in viral genome.
Detection method of the present invention, comprises the following steps:
1, design of primers: contain 6 kinds of HCoV that published before in June, 2013, comprising: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.According to human corona virus's complete sequence to be measured, select every kind of viral specific and conserved sequence, design PCR primer, 5 ' the sequence label of holding with 10 bases (ACGTTGGATG) arbitrary combination at primer, make total length reach 30 more than base, in order to primer and probe are differentiated from molecular weight.A single-basic extension probe of conserved sequence district design in amplification region, the length of probe is 14-28 base, 3 ' the end at probe, allow to extend one through designing definite base, as this Serotype-dependent sequence mark, after single-basic extension, total length is no more than 29 bases, and between the extension products of each probe, molecular weight differs at least 30Da.Various human corona virus's amplimer sequences are in Table 1, and its corresponding base extension probes is in Table 2.
2, PCR reaction: introduce uridylic N glycosylase (UNG enzyme) technology in PCR process, in PCR reaction system first, adopt the dTTP that substitutes conventional PCR with dUTP, make to mix in product a large amount of dU.Again carrying out before pcr amplification, with UNG enzyme, processing the residual contamination that PCR mixed solution can be eliminated PCR product.Because sex change one step of UNG enzyme in PCR circulation just can be inactivated, therefore can not affect new PCR reaction and product containing dU, thoroughly get rid of pcr amplification product and polluted the false positive problem causing.By first round pcr amplification, obtain the sample to be tested sequence amplification product that hits.
3, shrimp alkaline phosphotase (SAP) is processed: make unconjugated residue nucleic acid (dNTPs) dephosphorylation inactivation, pre-anti-tampering next step base extension.
4, base extension: the 3 ' end that carries out single-basic extension probe in second takes turns amplification, extend a sequence-specific mononucleotide, make molecular weight marker, the molecular weight difference between the extension products obtaining and described extension probes and each type extension products is not less than 16Da
5, resin desalination: purifying extension product.
6, mass spectrometric detection: adopt ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MASS) system to carry out molecular weight detection purifying after product, according to molecular weight marker, determine HCoV classification to be measured, every kind of viral detected result of the automatic mabage report of software and credibility.
Detection method of the present invention, preferably comprises the following steps:
(1), by one couple of PCR primers, the 5 ' sequence label of holding with the arbitrary combination of ACGTTGGATG base at primer, makes total length reach 30 more than base, to be different from probe; A single-basic extension probe of conserved sequence design in amplification region, the length of probe is 14-28 base, at 3 ' end of probe, allows to extend one through designing definite base, as this Serotype-dependent sequence mark, after single-basic extension, total length is no more than 29 bases;
(2) pcr amplification reaction for the first time, dUTP/dNTP mixture, UNG enzyme, Tag enzyme and multiple PCR primer are together added in PCR reaction system, first carry out the digestion of dUTP, degraded pcr amplification product, then deactivation UNG enzyme, do subsequently 45 cycle P CR amplification, obtain the sample to be tested sequence amplification product that hits;
(3) with shrimp alkaline phosphotase, process, make to remain dNTP dephosphorylation inactivation in first round reaction;
(4) single-basic extension amplification for the second time, adds ddNTPs in reaction system, makes it the 3 ' end at single-basic extension probe, extend a sequence-specific mononucleotide, make molecular weight marker, 200 circulations of increasing, between the extension products of each probe, molecular weight differs at least 16Da;
(5) adopt cationic exchange resin adsorption salt ion, purifying extension product;
(6) purifying after product adopts mass-spectrometric technique to carry out molecular weight detection, determines the type of HCoV to be measured according to molecular weight marker.
Each HCoV to be measured in wherein said step (1) design of primers, adopts distinguished sequence district.Primer sequence is SEQ ID NO.1 to SEQ ID NO.28, and probe sequence is SEQ ID NO.31 to SEQ ID NO.44.
In described step (1), preferably adopted sample quality to control reference, primer sequence is SEQ ID NO.29 and SEQ ID NO.30, and probe sequence is SEQ ID NO.45.
Preferably in every secondary response, add negative control, described negative control is deionization distilled water.
Method of the present invention, detectable level is low to moderate 10 copies/ul.
Table 1
Sequence numbering Primer sequence Human coronary virus
SEQ?ID?NO.1 ACGTTGGATGATGGGCTGATGMATCRGAAC HCoV-229E forward primer 1
SEQ?ID?NO.2 ACGTTGGATGCACTATCAACAAGCAAYGGG HCoV-229E reverse primer 1
SEQ?ID?NO.3 ACGTTGGATGTTTACTTGGCATGAGYGCAG HCoV-229E forward primer 2
SEQ?ID?NO.4 ACGTTGGATGGAGCGAAGCACAAATCRATC HCoV-229E reverse primer 2
SEQ?ID?NO.5 ACGTTGGATGGAAGCTACATGCAAAYCTGG HCoV-OC43 forward primer 1
SEO?ID?NO.6 ACGTTGGATGATGGATGTGGArACACYTCG HCoV-OC43 reverse primer 1
SEQ?ID?NO.7 ACGTTGGATGATTGCCAGAATTGGCRCTAC HCoV-OC43 forward primer 2
SEQ?ID?NO.8 ACGTTGGATGGAAGGTCTGCTCCTAYTTCC HCoV-OC43 reverse primer 2
SEQ?ID?NO.9 ACGTTGGATGGCACTTCTTTCAACTYATGG SARS-CoV forward primer 1
SEQ?ID?NO.10 ACGTTGGATGAGAGACACTCATAGAYCCTG SARS-CoV reverse primer 1
SEQ?ID?NO.11 ACGTTGGATGTCTTGGTTCACAGCTRTCAC SARS-CoV forward primer 2
SEQ?ID?NO.12 ACGTTGGATGCTGGACCACTATTGGTYTTG SARS-CoV reverse primer 2
SEQ?ID?NO.13 ACGTTGGATGTGTCAACCTGTACATTYCCC HCoV-NL63 forward primer 1
SEQ?ID?NO.14 ACGTTGGATGTGAGAACAAGTTTGTRCCTG HCoV-NL63 reverse primer 1
SEQ?ID?NO.15 ACGTTGGATGGCCAACGCTCTTGAYCATTC HCoV-NL63 forward primer 2
SEQ?ID?NO.16 ACGTTGGATGATTCCCAGGAATCTTYTCCC HCoV-NL63 reverse primer 2
SEQ?ID?NO.17 ACGTTGGATGGGAAACCTAGTAGGGARAGC HCoV-HKU1 forward primer 1
SEQ?ID?NO.18 ACGTTGGATGGCTAATCACCAAGCTGYCAC HCoV-HKU1 reverse primer 1
SEQ?IDNO.19 ACGTTGGATGCAATCAGCATACTCARAAAG Under HCoV-HKU1 to primer 2
SEO?IDNO.20 ACGTTGGATGTGCCACATATAGTTCYTAGG HCoV-HKU1 reverse primer 2
SEQ?ID?NO.21 ACGTTGGATGGCCACCRTAGAACTTRGTAG MERS-CoV forward primer 1
SEQ?ID?NO.22 ACGTTGGATGCTAATCGCCAGTACCARCAG MERS-CoV reverse primer 1
SEO?ID?NO.23 ACGTTGGATGTCCTCTTCACATAATRGCCC MERS-CoV forward primer 2
SEQ?ID?NO.24 ACGTTGGATGATGGATTAGCCTCTACRCGG MERS-CoV reverse primer 2
SEQ?ID?NO.25 ACGTTGGATGCATTACTCGTGAAGAYGCTG MERS-CoV forward primer 3
SE0?ID?NO.26 ACGTTGGATGATTACGGGAAGCATGYGCAC MERS-CoV reverse primer 3
SEQ?ID?NO.27 ACGTTGGATGTGTAGTTGGGATTCTTRGGG MERS-CoV forward primer 4
SEQ?ID?NO.28 ACGTTGGATGTGATGATCATGGCAACRCTG MERS-CoV reverse primer 4
SEQ?ID?NO.29 ACGTTGGATGAGATTTGGACCTGCGAGRGG Quality Control forward primer
SEQ?ID?NO.30 ACGTTGGATGTGAGCGGCTGTCTCCARAAG Quality Control reverse primer
Table 2.
Sequence numbering HCoV Base extension probes sequence
SEQ?ID?NO.31 HCoV-229E probe 1 GACACGGGTATTCTACCCTG
SEQ?ID?NO.32 HCoV-229E probe 2 ATTACCATCCTGTCTACTAAC
SEQ?ID?NO.33 HCoV-OC43 probe 1 CGTGGATACACATCGTTAT
SEQ?ID?NO.34 HCoV-OC43 probe 2 CGCTCCTAATTCCAGATCTACTT
SEQ?ID?NO.35 SARS-CoV probe 1 GATTGGCCTGTGTTGTA
SEQ?ID?NO.36 SARS-CoV probe 2 CTTGAGGGAATCTAAGTTCCTC
SEQ?ID?NO.37 HCoV-NL63 probe 1 TAAACCGTTTGTCCCTGT
SEQ?ID?NO.38 HCoV-NL63 probe 2 TTTGGCAGGAATCTTGTCCCTAT
SEQ?ID?NO.39 HCoV-HKU1 probe 1 CTCCGCTGACACTTCTA
SEQ?ID?NO.40 HCoV-HKU1 probe 2 AGCATTGGTTAGATCTTTGCTATG
SEQ?ID?NO-41 MERS-CoV probe 1 TCGATGGCTGCAAC
SEQ?ID?NO.42 MERS-CoV probe 2 CTTAAACGCAGAGCTG
SEQ?ID?NO.43 MERS-CoV probe 3 CGCCCATTGAGCACCCTCAA
SEQ?ID?NO.44 MERS-CoV probe 4 CCCCCGCCCTGTGTACTTCCTTC
SEQ?ID?NO.45 RNA enzyme P gene probe CTTCCGCGCAGAGC
Table 1, the synthetic polynucleotide of listing in table 2, all adopt conventional polynucleotide synthetic method synthetic.
Compare with similar technology with other technology of existing detection HCoV, technical solution of the present invention has been given full play to PCR-mass spectrometry and has been detected viral advantage, to 15 heavy PCR reaction product, make disposable mass spectrometric detection, do not need fluorochrome label, do not need washing, react and carry out in micro-system, reagent consumables cost is inversely proportional to detecting PCR tuple, thereby realizes with many probes, in a reaction, detect the target of 6 kinds of HCoV simultaneously.The Serotype-dependent fragment that first run pcr amplification region is 60-120bp, second takes turns PCR adopts the amplification of specific probe end one-point method, can allow to use more circulations, detection sensitivity and specificity have been improved to greatest extent, make detection sensitivity reach the single copy level of 1aM level, avoided detecting and failed to pinpoint a disease in diagnosis the Problem of False Negative causing.Adopt UNG zymotechnic and Serotype-dependent fragment amplification designing technique simultaneously, thoroughly got rid of the false positive problem that PCR product pollution and the mispairing of homologous sequence probe cause.For HCoV pathogenesis provides reliable experimental technique.
According to detection method of the present invention, the inventor has also designed a kind of detection kit, and to the detection for clinical sample, test kit of the present invention, can comprise that one or more are as table 1, the synthetic polynucleotide reagent of table 2.
Test kit of the present invention comprises following reagent:
Synthetic polynucleotide reagent, described polynucleotide are forward and the reverse primer sequence SEQ ID NO.1-SEQ ID NO.28 of 6 kinds of HCoV;
Base extension probes sequence SEQ ID NO.31-SEQ ID NO.44;
Quality Control primer sequence is SEQ ID NO.29 and SEQ ID NO.30 and probe sequence SEQ ID NO.45.
As required, in test kit of the present invention, can also include the reagent that is beneficial to laboratory operation as solvent, buffer solvent, subsidiary material etc.
Test kit of the present invention, can comprise the reagent that above component is prepared from, and the compound method of reagent is routine techniques, only various starting material need to be mixed at normal temperatures, without specific installation and condition.
Test kit of the present invention, can distinguish splendid attires by different reagent, more together be packaged in same packing box, during use, according to the method for describing in specification sheets, operates.
Accompanying drawing explanation
Fig. 1,15-Plex human corona virus base extension probes mass spectra peak figure
Embodiment
The present invention includes and detect one or more in 6 kinds of known HCoV simultaneously, for HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV is used 2 heavily to react and detect for distinguished sequence district, for MERS-CoV, for distinguished sequence district, uses 4 heavily to react and detect.Embodiment described herein only, for explaining the present invention, is not intended to limit the present invention.
The high-sensitivity detection that embodiment of the present invention provides and/or evaluation human coronary virus's method, 6 kinds of human corona viruses to be detected comprise: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.Comprise the steps:
(1) according to HCoV whole genome sequence to be measured, select the specific and conserved sequence of every kind of HCoV, design PCR primer, 5 ' the sequence label of holding with 10 bases (ACGTTGGATG) arbitrary combination at primer, make total length reach 30 more than base, in order to primer and probe are differentiated from molecular weight.The amplimer sequence of various HCoV is in Table 1.Conserved sequence district in amplification region, design a single-basic extension probe, the length of probe is 14-28 base, 3 ' the end at probe, allow to extend one through designing definite base, as this Serotype-dependent sequence mark, after single-basic extension, total length is no more than 29 bases, and between the extension products of each probe, molecular weight differs at least 30Da.Various HCoV probe primer sequences are in Table 2.
(2), by pcr amplification, obtain the sample to be tested sequence amplification product that hits.
The preparation of PCR reaction system is in Table 3.
Table 3
Reagent Ultimate density Volume (1 *)
Water,HPLC?grade N/A 0.3μL
10X?Buffer?w/20mM?MgC1 2 2mM?MgC1 2 0.5μL
25mM?MgCl 2 2mM 0.4μL
25mM?dUTP/dNTP?Mix 500mM 0.lμL
UNG?Enzyme(1U/ul) 0.5U 0.5μL
0.5mM?Primer?mix 0.1mM 1.0μL
5U/mlPCR?enzymea 1Unit 0.2μL
Template?DNA(5-20ng/uL) ? 2.0μL
Total ? 5.0μL
First use 37 ℃-50 ℃ 2 minutes, carry out the digestion of dUTP, then 94 ℃ of deactivations in 4 minutes, (simultaneously this step also reaches the effect of denaturation), makes pcr amplification subsequently.Reaction conditions is 95 ℃ of sex change 30 seconds, anneals 30 seconds for 56 ℃, and 72 ℃ are extended 1 minute, totally 45 circulations; Final 72 ℃ are extended 5 minutes, and after completing, constant temperature is in 4 ℃.
(3) by shrimp alkaline phosphotase (SAP), process, make unconjugated residue nucleic acid (dNTPs) dephosphorylation inactivation, pre-anti-tampering next step base extension.SAP digestive ferment reaction system is in Table 4.
Table 4
Reagent Volume/reaction
Water,HPLC?grade 1.53μL
10xSAP?buffer 0.17μL
SAP?enzyme(1.7U/ml) 0.3μL
Total 2.0μL
Reaction conditions is 37 ℃ hatches 40 minutes, removes residue dNTPs; Then with 85 ℃, within 5 minutes, make SAP enzyme deactivation.
(4) by base extension, 3 ' the end at single-basic extension probe, extends a sequence-specific mononucleotide, makes molecular weight marker, molecular weight difference between the extension products obtaining and described extension probes and each type extension products is not less than 16Da, and extension system is in Table 5.
Table 5
Reagent Volume/reaction
Water,HPLC?grade 0.619μL
10×iPLEX?buffer?plus 0.2μL
iPLEX?terminator?mix 0.2μL
Primer?mix* 0.94μL
iPLEX?enzyme 0.041μL
Total 2.0μL
* the concentration of extension probe mix, carries out linear relationship adjusting according to various molecular size range, and total concn is about 8-15M, and ultimate density is about 0.84-1.57M.
Circulating reaction is 200 short ladder programs, comprises two circulations chimeric, and starting is 94 ℃ of sex change 30 seconds, subsequently 94 ℃ 5 seconds, 52 ℃ of annealing 5 seconds, 80 ℃ are extended 5 seconds, in totally 40 circulations, each inserts annealing and extends 5 partial circulatings; Final 72 ℃ are extended 3 minutes.
(5) adopt resin desalting and purifying extension product.
(6) purifying after product is adopted ground substance assistant laser desorption ionization flight time mass spectrum system carry out molecular weight detection, according to molecular weight marker, determine HCoV classification to be measured.In Fig. 1, X-coordinate is molecular weight, and ordinate zou is peak intensity.In figure stringer dotted line homochromy be the molecular weight position of this genotype extension probes, if there is no this type probe peak is constant; If be detected HCoV copy number, be greater than 10, probe can be by completely consumed, and peak, left side disappears and goes to right side; The homochromy dotted line on right side represents extension products molecular weight position.With TYPE4.0 software, read spectrogram, automatic analysis and reporting the result, derived data.Data interpretation is, the extension probes of each HCoV and reference gene has corresponding molecular weight peaks at the different quality place of mass spectrum, when probe is found target gene work, occur that single-basic extension product, probe molecule amount peak occur to shift for molecular weight of product peak, analysis result information is positive.Positive findings is divided into four kinds: A, reliable results; B, moderate are reliable; C, generally reliable; D, low reliable.It is effective that first three kind is considered as extension, has corresponding HCoV to infect, and whether the 4th kind of needs human assistance judgement, observe probe and consume, and is judged as suspicious taint or negative findings.To suspicious taint sample, can make repeated proof test where necessary.

Claims (1)

1. the test kit that can simultaneously detect 6 kinds of human corona viruses, it is characterized in that, the polynucleotide that comprise SEQ ID NO.1-SEQ ID NO.45 in described test kit, wherein, SEQ ID NO.1-SEQ ID NO.28 is 6 kinds of human corona viruses' forward and reverse primer sequence; SEQ ID NO.31-SEQ ID NO.44 is base extension probes sequence; SEQ ID NO.29 and SEQ ID NO.30 are Quality Control primer sequence; SEQ ID NO.45 is Quality Control base extension probes sequence,
Wherein, described human corona virus comprises: human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV).
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