CN1570139A - Atypical pneumonia coronary virus gene detecting method and kit - Google Patents
Atypical pneumonia coronary virus gene detecting method and kit Download PDFInfo
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- CN1570139A CN1570139A CNA031418392A CN03141839A CN1570139A CN 1570139 A CN1570139 A CN 1570139A CN A031418392 A CNA031418392 A CN A031418392A CN 03141839 A CN03141839 A CN 03141839A CN 1570139 A CN1570139 A CN 1570139A
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Abstract
The invention provides a sample concentration, RT-PCR amplification, and product detecting method for detecting the SARS coronavirus gene and its kit. The method comprises: (a) concentrating sample by using PEG precipitation method; (b) extracting the virus PNA in the sample, and fluorescent RT-PCR amplification by using specific primer; (c) fluorescent quantitative detection for the amplified product; (d) electrophoresis for the PCR product in step (c). According to the method, the existence of the specific amplified product shows that there is the SARS coronavirus in the sample.
Description
Technical field
The present invention relates to the technique of gene detection field, relate more specifically to the gene tester and the test kit of SARS coronavirus.
Background technology
China in 2003 and many in the world countries have suffered the once invasion and attack of large-scale atypical pneumonia (Atypicalpneumonias), (Severe AcuteRespiratory Sydrome SARS) takes place 22 countries and regions the severe acute respiratory syndrome that is caused by atypical pneumonia.(WorldHealth Organization WHO) sets up cooperating research and the monitoring network of being made up of 13 laboratories of 10 countries in the whole world rapidly after epidemic situation takes place in the World Health Organization.April 16, the pathogenic agent of WHO official confirmation SARS is a kind of novel coronavirus, and this is a kind of single strand RNA virus.
The existing 8000 many cases atypical pneumonia patients in the whole world, its mortality ratio reaches 15% in some region of disease, has brought great fear to the people.In time carrying out the etiology of sars coronavirus and the research of prevention, diagnosis and treatment, tighten control and the treatment measure, patient and suspected case are accomplished early to find, early isolated and early treatment, is that the control epidemic situation continues the key that takes place and propagate.
After the human infection SARS virus, through producing corresponding antibody behind the certain hour, can detect antibody in the serum by antigen-antibody reaction.Can detect specific antibody with methods such as immunofluorescent test and enzyme linked immunosorbent assay at SARS virus.It is later that but this antibody occurs, and generally will arrive and infect back about 20 days, is unfavorable for the early diagnosis of virus infection.
The most direct method of diagnosis SARS is to detect the intravital pathogenic agent of patient, and (Polymerase Chain Reaction PCR) detects viral genetic material can to utilize the polymerase chain reaction.Reverse transcription polymerase chain reaction (RT-PCR) at first becomes DNA with a spot of SARS virus RNA reverse transcription in the sample, by gene amplification nucleic acid is amplified millions of times again, can detect by electrophoresis or fluorescent quantitation, be applied to the nucleic acid of SARS virus in infecting early stage (in 10 days) human body respiratory secretions, blood, serum, urine, ight soil or dead patient's necrotomy sample, have easy fast, characteristics such as high specificity.
Yet because in the biological sample that obtains, the content of SARS virus is considerably less, and blood, ight soil, respiratory secretions wait for that test sample product also contain other nucleic acid substances in a large number.Therefore, this has brought great difficulty for the SARS specificity product that effectively increases, and often causes false-negative result in experiment.
Therefore, this area presses for the new technology that detects SARS virus quickly and accurately of exploitation.
Summary of the invention
Purpose of the present invention just provides a kind of novel method and related kit that detects SARS virus quickly and accurately.
In a first aspect of the present invention, the concentration method of SARS virus in a kind of sample is provided, more preferably concentrate with PEG.Be 5000-25000 dalton with molecular weight for example, the PEG as 6000,8000,20000 concentrates, and especially molecular weight is that 20000 PEG concentrates.
In a second aspect of the present invention, a kind of method of SARS coronavirus specific amplification is provided, it comprises the steps:
(a) viral RNA in the extracting sample;
(b), under the specific amplification condition, carry out the fluorescence RT-PCR amplification with Auele Specific Primer shown in the SEQ ID NO:1 and 2 and the nucleic acid in the step (a).
In a third aspect of the present invention, a kind of method that specific amplification products is detected is provided, it comprises the steps:
(c) product to step (b) carries out the fluorescent quantitation detection;
(d) to the amplified production of the negative step (b) of part fluorescent quantitation detected result, the Auele Specific Primer with shown in the SEQ IDNO:3 and 4 carries out the pcr amplification second time under specific amplification condition;
(e) the PCR product to step (d) carries out electrophoresis, shows specific band and just represents to exist in the sample SARS coronavirus.
In a fourth aspect of the present invention, a kind of test kit that detects SARS coronavirus is provided, it contains the Taqman probe of nucleotide sequence shown in the primer, SEQID NO:5 of nucleotide sequence shown in the reagent that concentrates sample, the SEQ ID NO:1 and 2.Described test kit also contains the required reagent of preparation template, carries out the required reagent of pcr amplification.
In another preference, described test kit also contains the nest-type PRC primer of the nucleotide sequence shown in SEQ ID NO:3 and 4.
Description of drawings
Fig. 1 has shown the quantitative fluorescent PCR curve of each sample among the embodiment 3.
Fig. 2 has shown electrophoresis detection figure.Wherein: swimming lane 1 negative contrast; Swimming lane 2 positive contrasts; Swimming lane 3,4 is sample No. 5; Swimming lane 5 is the molecular weight standard thing.
Embodiment
The inventor is extensive studies through going deep into, at first utilize the virus in the concentrated sample of the PEG precipitator method, the SARS Auele Specific Primer has been synthesized in design, pass through fluorescence quantitative RT-RCR, in conjunction with nest-type PRC, realize the early detection fast and accurately of SARS virus, finished the present invention on this basis.
Particularly, the inventor according to different areas such as Hong Kong, Guangzhou, Beijing, Toronto the genome sequence of isolating coronavirus strain, 157 bases having selected CUHK-W1 strain P gene (polymerase protein of coding virus) 18138bp-18294bp are as target nucleotide, and its sequence is as follows:
atgaattacc?aagtcaatgg?ttaccctaat?atgtttatca?cccgcgaaga?agctattcgt????60
cacgttcgtg?cgtggattgg?ctttgatgta?gagggctgtc?atgcaactag?agatgctgtg????120
ggtactaacc?tacctctcca?gctaggattt?tctacag?????????????????????????????157
(SEQ?ID?NO：6)
To the result of above 157 base Blast in Genebank as can be known, this target sequence conservative property is very high, and except that the Guangzhou strain had the difference of a base, other sequence was homology.
According to the sequence shown in the SEQID NO:6, the present invention designs and has synthesized the primer of following RT-PCR:
SARS-Fs：5′-ATG?AAT?TAC?CAA?GTC?AAT?GGT?TAC-3′(SEQ?ID?NO：1)
SARS-Fas：5′-CTG?TAG?AAA?ATC?CTA?GCT?GGA?G-3′(SEQ?ID?NO：2)
Real-time fluorescence quantitative PCR is a new developing technology on the PCR basis, and it has utilized 5 ' → 3 ' 5 prime excision enzyme activity of Taq enzyme, adds a fluorescence labeling probe in the PCR reactive system, can carry out accurately quantitatively template.A kind of sequence of preferred TaqMan probe is as follows:
5′-FAM-TCG?TGC?GTG?GAT?TGG?CTT?TGA?TGT-TAMRA-3′(SEQ?ID?NO：5)
Wherein, fluorescent emission group and fluorescent quenching group can adopt this area kind commonly used, and for example the fluorescent emission group can adopt 6-Fluoresceincarboxylic acid (FAM), and the fluorescent quenching group can adopt 6-carboxyl tetramethylrhodamin (TAMRA).
For the negative suspicious specimen of fluorescent quantitation detected result, the fluorescence RT-PCR product can be carried out the amplification second time.In detection method of the present invention, it can effectively improve the sensitivity of detection.
In the present invention, the sequence of SARS coronavirus specificity nested primer is corresponding to the nucleotide sequence shown in the SEQ IDNO:6, and length is 18-30bp (preferably 19-25bp).A kind of primer of preferred nest-type PRC is as follows:
SARS-ins：5-GAA?GCT?ATT?CGT?CAC?GTT?CG-3(SEQ?ID?NO：3)
SARS-Fas：5-CTG?TAG?AAA?ATC?CTA?GCT?GGA?G-3(SEQ?ID?NO：4)
When the nested primer shown in the use SEQ ID NO:3 and 4, the product after the amplification has the sample of specific band can be judged to positive findings, otherwise is judged to negative findings through agarose gel electrophoresis at the 109bp place.Adopt aforesaid method to help the early diagnosis of atypical pneumonia sars coronavirus.Detect fast, can get the result in general 4 hours.
In the methods of the invention, to the not special restriction of pcr amplification condition, can adopt the specific PCR amplification condition of this area routine.
In the methods of the invention, the extraction of RNA is very important.Because contained viral load is less in some sample, in detecting, PCR causes false negative easily.A kind of preferable methods is to come coronavirus in the enriched sample with the method for PEG precipitated rna.The sample throw out carries out the fluorescence RT-PCR amplification under the effect of Auele Specific Primer, SuperscriptII reversed transcriptive enzyme, DNA Taq enzyme, fluorescence labeling probe etc. after the RNA extracting.
The present invention also provides a kind of test kit that detects SARS coronavirus, and it contains the Taqman probe of nucleotide sequence shown in the primer, SEQ ID NO:5 of nucleotide sequence shown in the reagent that concentrates sample, the SEQ ID NO:1 and 2.Described test kit also contains the required reagent of preparation template, carries out the required reagent of pcr amplification.
As used herein, " the required reagent of preparation template " refers to that the RNA that will detect in the sample extracts the used reagent of purge process.For example: Qiagen company viral RNA extraction agent.
As used herein, " carrying out the required reagent of pcr amplification " refers to the denier SARS coronavirus gene in the template is carried out the used reagent of pcr amplification reaction process, comprising for example: dNTP, high temperature-resisting DNA polymerase, Mg
2+, Auele Specific Primer and damping fluid etc.
Major advantage of the present invention is:
(a) sample is carried out the PEG concentration, increased the quantity of virus in the sample.
(b) product detects and adopts fluorescent quantitation, and combines with nest-type PRC, has improved detection sensitivity.
Fluorescent quantitation sensitivity: 10
3Copy/ml; Nest-type PRC sensitivity: 10
2Copy/ml.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Concentrating of sample:
Get in the centrifuge tube that 20%PEG solution 170ul adds 1.5ml, add sample 500ul, add 10.2ul concentration again and be 20% sulfuric acid, vibration mixing 5s; 4 ℃ of placements are spent the night, 4 ℃ of centrifugal 17860g, 20min; Abandoning supernatant, 4 ℃ of centrifugal 17860g, 1min.Remove unnecessary supernatant liquor with careful suction of adjustable pipette, precipitation is used for the sample extracting.
Embodiment 2
Qiagen company viral RNA extraction agent box is adopted in the sample extracting, operates by the specification sheets that manufacturers provides.
Embodiment 3
The fluorescence RT-PCR amplification
Primer adopts:
SARS-Fs：5-ATG，AAT，TAC，CAA，GTC，AAT，GGT，TAC-3(SEQ?ID?NO：1)
SARS-Fas：5-CTG，TAG，AAA，ATC，CTA，GCT，GGA，G-3(SEQ?ID?NO：2)
The TaqMan probe adopts:
5′-FAM-TCG，TGC，GTG，GAT，TGG，CTT，TGA，TGT-TAMRA-3′(SEQ?ID?NO：5)
The RT-PCR amplifing reagent:
RT-PCR main reaction liquid (contains primer, dNTP/dUTP, probe, Mg
2+Deng)
RT-PCR enzyme mixed solution (solution that contains Superscript II reversed transcriptive enzyme, Taq enzyme and RNA enzyme inhibitors)
Uridylic-N-glycosylase (UNG)
Operation: with briefly centrifugal behind the thawing of 33.8ul RT-PCR main reaction liquid, the mixing, with 0.8ulRT-PCR enzyme mixed solution, 0.4ul UNG thorough mixing, in reaction tubes, add sample filtrate and the working standard of having handled well respectively, cover lid is put into iCycler (Roche company) and is increased after the centrifugal several seconds.
The result obtains: select fluorophor FAM-490, obtain the fluorescence result, shown in Fig. 1 and table 1.
Table 1 fluorescence RT-PCR detected result
| The sample sequence number | ??1 | ??2 | ??3 | ??4 | ??5 |
| The Ct value | ??22.8 | ??N/A | ??26.9 | ??32.0 | ??N/A |
| Judged result | ??+ | ??- | ??+ | ??+ | Suspicious |
Ct value: cycle threshold
N/A: do not detect signal
+: judged result is positive
-: judged result is negative
Sample is provided with: 1 is the positive reference material of RNA; 2 is normal people's negative serum; 3,4 is the SARS patient's of Shanghai 2 routine clinical conclusive evidences serum.Mark this shop 5 is the suspicious patient's of certain SARS a serum.
This result shows that 3, No. 4 detection result of specimen is positive, though No. 5 sample fluorescent quantitation detected results are N/A, in conjunction with its fluorescent quantitation detection curve, the result is suspicious.
Embodiment 4
Nest-type PRC
For the negative suspicious specimen of fluorescent quantitation detected result among the embodiment 3, fluorescence PCR products can be carried out nest-type PRC, the primer of nest-type PRC adopts:
SARS-ins：5-GAA?GCT?ATT?CGT?CAC?GTT?CG-3(SEQ?ID?NO：3)
SARS-Fas：5-CTG?TAG?AAA?ATC?CTA?GCT?GGA?G-3(SEQ?ID?NO：4)
Pcr amplification reagent:
2
NdPCR Taq polysaccharase
2
NdThe PCR reaction solution (contains primer, dNTP/dUTP, Mg
2+Deng)
UNG
Operation: with 27.4ul 2
NdThe PCR reaction solution melts, and is briefly centrifugal behind the mixing, with 0.3ul 2
NdPCR Taq polysaccharase, 0.3ul UNG thorough mixing add suspicious fluorescence PCR products 2ul in reaction tubes, put into the PCR instrument and increase.
The result: as shown in Figure 2, No. 5 sample has specific band at the 109bp place, is the SARS positive.
Embodiment 5
The SARS coronavirus detection kit
For convenient above-mentioned PCR detects, the gene detecting kit of a kind of SARS coronavirus of special one-tenth, this test kit comprises following reagent:
(1) sample virus enrichment reagent:
20%PEG solution (being dissolved in 0.9%NaCl solution)
(2) viral RNA extraction agent box (Qiagen company)
(3) RT-PCR reagent
RT-PCR main reaction liquid:
1 * damping fluid
2.5mM?MgCl
2
0.2uM primer SARS-Fs (SEQ ID NO:1)
0.2uM primer SARS-Fas (SEQ ID NO:2)
0.2mM?dNTP
0.2mM?dUTP
0.1uM probe
RT-PCR enzyme mixed solution:
2U/ul Superscript II reversed transcriptive enzyme
0.05U/ul Taq archaeal dna polymerase
0.01U/ul?UNG
(4) nest-type PRC reagent
Reaction solution:
1 * damping fluid
2.5mM?MgCl
2
0.2uM primer SARS-ins (SEQ ID NO:3)
0.2uM primer SARS-Fas (SEQ ID NO:4)
0.2mM?dNTP
0.2mM?dUTP
0.05U/ul 2
NdPCR Taq polysaccharase
0.01U/ul?UNG
(5) electrophoresis reagent
50 * electrophoretic buffer
The tetrabromophenol sulfonphthalein damping fluid
2% gel
Ethidium bromide
Positive object of reference.
Embodiment 6
Concentrate influence to detected result
With the PEG of different molecular weight, test sample is concentrated according to the method for embodiment 1.And carry out the amplification of RNA extracting, fluorescence RT-PCR by the method for embodiment 2-4.With without crossing concentration, but the detected result of carrying out extracting, fluorescence RT-PCR amplification equally compares.
The result is as shown in the table:
| Negative control | Standard substance 1 | Standard substance 2 | Standard substance 3 | Sample stoste | ??PEG ??20000 | ??PEG ??8000 | ??PEG ??6000 | |
| The CT value | ??- | ??30.2 | ??26.5 | ??22.8 | ??32.88 | ??24.3 | ??26.6 | ??26.3 |
| Quantitatively (copy number/ml) | ??0 | ??50 | ??500 | ??5000 | ??4.27×10 2 | ??2.07×10 5 | ??4.81×10 4 | ??5.80×10 4 |
Wherein, standard substance 1, standard substance 2, standard substance 3 are used to the curve that settles the standard.Measured value without the sample stoste of concentration is 4.27 * 10
2Copy/ml, and with after PEG20000, PEG8000 and the PEG6000 concentration, measured value brings up to 2.07 * 10
5, 4.81 * 10
4, and 5.80 * 10
4Copy/ml is wherein especially with concentrated effect the best of PEG20000.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Fudan-Yueda Bio-Tech Co., Ltd.
＜120〉gene tester of SARS coronavirus and test kit
<130>033384
<160>6
<170>PatentIn?version?3.1
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(24)
＜223〉primer
<400>1
atgaattacc?aagtcaatgg?ttac?????????????????????????????????????????24
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primer
<400>2
ctgtagaaaa?tcctagctgg?ag???????????????????????????????????????????22
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(20)
＜223〉primer
<400>3
gaagctattc?gtcacgttcg??????????????????????????????????????????????20
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(23)
＜223〉primer
<400>4
ctgtagaaaa?tcctagctgg?ag???????????????????????????????????????????22
<210>5
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(24)
＜223〉probe
<400>5
tcgtgcgtgg?attggctttg?atgt?????????????????????????????????????????24
<210>6
<211>157
<212>DNA
＜213〉atypical pneumonia virus (SARS virus)
<400>6
atgaattacc?aagtcaatgg?ttaccctaat?atgtttatca?cccgcgaaga?agctattcgt??60
cacgttcgtg?cgtggattgg?ctttgatgta?gagggctgtc?atgcaactag?agatgctgtg??120
ggtactaacc?tacctctcca?gctaggattt?tctacag???????????????????????????157
Claims (10)
1. whether there is the method for SARS coronavirus in the test sample, it is characterized in that, comprise step:
(a) using the PEG precipitator method concentrates sample;
(b) viral RNA in the extracting sample carries out the fluorescence RT-PCR amplification with Auele Specific Primer under specific amplification condition;
(c) amplified production is carried out fluorescent quantitation and detect,, carry out the pcr amplification second time with the specificity nested primer to the negative suspicious sample of part fluorescent quantitation detected result;
(d) the PCR product to step (c) carries out electrophoresis, exists specific amplification products just to represent to exist in the sample SARS coronavirus.
2. the method for claim 1 is characterized in that, the primer of described fluorescence RT-PCR has the nucleotide sequence shown in SEQID NO:1 and 2.
3. the method for claim 1 is characterized in that, described nested primer has the nucleotide sequence shown in SEQ ID NO:3 and 4.
4. the method for claim 1 is characterized in that, uses the PEG of molecular weight as 5000-25000 in the PEG precipitator method.
5. a test kit that detects SARS coronavirus is characterized in that, it contains the primer of nucleotide sequence shown in SEQ ID NO:1 and 2.
6. test kit as claimed in claim 5 is characterized in that, also contains the nested primer of nucleotide sequence shown in SEQ ID NO:3 and 4.
7. test kit as claimed in claim 5 is characterized in that, also contains the required reagent of preparation template, carries out the required reagent of pcr amplification.
8. test kit as claimed in claim 5 is characterized in that, also contains the Taqman probe of nucleotide sequence shown in the SEQ ID NO:5.
9. test kit as claimed in claim 5 is characterized in that, also contains the PEG reagent that concentrates sample virus.
10. test kit as claimed in claim 5 is characterized in that, the molecular weight of described PEG is 5000-25000.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031418392A CN1570139A (en) | 2003-07-25 | 2003-07-25 | Atypical pneumonia coronary virus gene detecting method and kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031418392A CN1570139A (en) | 2003-07-25 | 2003-07-25 | Atypical pneumonia coronary virus gene detecting method and kit |
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| Publication Number | Publication Date |
|---|---|
| CN1570139A true CN1570139A (en) | 2005-01-26 |
Family
ID=34471071
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|---|---|---|---|
| CNA031418392A Pending CN1570139A (en) | 2003-07-25 | 2003-07-25 | Atypical pneumonia coronary virus gene detecting method and kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086021B (en) * | 2007-06-08 | 2010-05-26 | 哈尔滨工业大学 | Primer for detecting sulfate reduction bacterium |
| CN111676325A (en) * | 2020-07-07 | 2020-09-18 | 云南科耀生物科技有限公司 | Primer combination for detecting SARS-CoV-2 whole genome and application method |
| CN111876523A (en) * | 2020-01-15 | 2020-11-03 | 福州大学 | One-step kit for molecular detection of coronavirus |
-
2003
- 2003-07-25 CN CNA031418392A patent/CN1570139A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086021B (en) * | 2007-06-08 | 2010-05-26 | 哈尔滨工业大学 | Primer for detecting sulfate reduction bacterium |
| CN111876523A (en) * | 2020-01-15 | 2020-11-03 | 福州大学 | One-step kit for molecular detection of coronavirus |
| CN111876523B (en) * | 2020-01-15 | 2021-07-13 | 福州大学 | One-step method kit for molecular detection of SARS-CoV-2 coronavirus |
| CN111676325A (en) * | 2020-07-07 | 2020-09-18 | 云南科耀生物科技有限公司 | Primer combination for detecting SARS-CoV-2 whole genome and application method |
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