CN101760562A - Hepatitis A virus detecting method and kit thereof by nucleic acid isothermal amplication - Google Patents
Hepatitis A virus detecting method and kit thereof by nucleic acid isothermal amplication Download PDFInfo
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- CN101760562A CN101760562A CN200810201681A CN200810201681A CN101760562A CN 101760562 A CN101760562 A CN 101760562A CN 200810201681 A CN200810201681 A CN 200810201681A CN 200810201681 A CN200810201681 A CN 200810201681A CN 101760562 A CN101760562 A CN 101760562A
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Abstract
The present invention relates to a hepatitis A virus (HAV) or attenuated vaccine detecting method and a kit thereof by nucleic acid isothermal amplication. The detection includes the steps in sequence of unwinding the RNA template of HAV under 60 to 70 DEG C and then cooling, adding with reverse transcriptase, ribonuclease H, RNA polymerase, performing the isothermal amplication under 37 to 42 DEG C conducted by primer to obtain RNA amplicon, then detecting the RNA amplicon by gold nanometer probe and capture probe, and obtaining the detecting result through chromatography cross-chromogenic reaction. The RNA is positive if chromogenic stripe appears on the cross film and the RNA is negative or else. The kit of the method can detect the RNA of HAV and attenuated vaccine quickly and accurately.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the RNA augmentation detection technology of hepatitis A virus (HAV) or attenuated vaccine.
Background technology
Hepatitis A virus (Hepatitis A virus, HAV is called for short hepatitis A) be the pathogenic agent of this transmissible disease of Hepatitis A, it belongs to Pironavirus, genome is the positive chain RNA that a treaty contains 7400 Nucleotide, and virion is twenty body structure, the about 27～32nm of size.Hepatitis A virus (HAV) is mainly propagated through fecal-oral route, and the pollution of drinking-water causes hepatitis A popular major cause often, and the HAV infection rate is about 50～70% in China adult; Show that according to Ministry of Health's announcement data national hepatitis A sickness rate is about 16,/10 ten thousand, hepatitis A is as a kind of Pandemic infection disease simultaneously, and about 4,000,000,000 people in the whole world are subjected to its threat.Can hole up in the body behind the HAV virus infection 14～45 days, its clinical symptom comprises heating, tired, nauseating apocleisis and abdominal discomfort etc., and jaundice can appear in 2/3 infection adult simultaneously, and may cause people's death, mortality ratio when the HAV acute infection is 1～3 ‰.In recent years, because the increase of movement of population, accelerated the spread scope and the speed of hepatitis A in China, characteristics of incidence present virus infection to the big age passes, Susceptible population constantly accumulates, the popular dangerous increase of hepatitis A.The main means of control and prevention hepatitis A popular are to use Attenuated HAV to carry out the immunization prevention at present.
Except the prevention and control of HAV, also need a kind of sensitivity fast detection method be used for the diagnosis of HAV virus.The detection method of hepatitis A virus (HAV) mainly contains virus culture, antigen or antibody test at present, RNA detects four kinds.Virus culture need be seeded on the sensitive cells, but because poor growth needs 3～4 time-of-weeks, therefore is not used in clinical HAV routine diagnosis.Antigen or antibody test are used in clinical detection extensively, but since on the methodology antibody need behind virus infection for some time to occur and there is nonspecific reaction in antigen-antibody, influenced the sensitivity and the specificity that detect.It mainly is by reverse transcription polymerase chain reaction (Reverse transcription-Polymerase Chain Reaction that HAV RNA detects, RT-PCR) technology realizes, the particularly development of fluorescent RT-PCR technology has in recent years promoted the progress that HAV RNA detects.Though it is very high that fluorescent RT-PCR technology detects HAV RNA sensitivity, need the temperature cycling equipment of complex and expensive when using, and higher to operator's technical requirements, limited the large-scale promotion of this technology and used.(Nucleic Acid Isothermaland Transcriptional Amplification with Gold Nano-particle Probes NITAG) is a kind of novel nucleic acids detection technique (principle is referring to accompanying drawing 1) of utilizing Nano-Au probe rapid detection constant-temperature amplification product for nucleic acid isothermal transcription amplification and Nano-Au probe detection method.Particularly, the reversed transcriptive enzyme that the NITAG technology is at first utilized a kind of multienzyme complex and had, the rna polymerase activity of RNase H and dependence DNA, at a large amount of RNA amplicons of the nucleic acid-templated acquisition of external isothermal duplication, target sequence hybridization among the oligonucleotide probe of nano gold mark and these RNA forms the poly network structure of the nm gold particles-polynucleotide that stretch then, cause the variation of nano Au particle optical property thus, producing hybridization signal shows with hybridization solution/plate/test paper colour-change, exist interactional hybridization solution/plate/test paper to be shown as blueness, effect is not shown in red.Utilize this technology, the color signal twin-stage amplification that micro-RNA template can detect by the template copy number and the Nano-Au probe of isothermal duplication obtains detecting, and this detection technique has advantages such as the special expensive of need not equipment, detection is sensitive fast, visual result is visible.Adopt the application of NITAG technology for detection HAV RNA still not have open report at present, can fast and effeciently diagnose hepatitis A virus (HAV) or its attenuated vaccine, have accurately sensitive, the fast and convenient characteristics that detect by detecting.
Summary of the invention
Technical problem to be solved
Technical problem to be solved by this invention provides a kind of hepatitis A virus (HAV) or attenuated vaccine RNA detection method and test kit based on the NATAG technology, and the RNA from HAV is carried out isothermal duplication and Nano-Au probe detection.
Technical scheme
The invention provides a kind of 5 ' end non-coding region based on NITAG technology amplification hepatitis A virus (HAV) or attenuated vaccine RNA (5 '-UTR) oligonucleotide primer to and probe, wherein primer is to sequence with SEQ ID NO:1 and SEQ ID NO:2 (SEQ ID NO:25 ' end contains the promoter sequence that can be discerned by t7 rna polymerase); Detection probes has the sequence (the former 5 ' end and the process hexane sulfhydrylation processing respectively of the latter's 3 ' end) of SEQ ID NO:3 and SEQ ID NO:4, and capture probe has the sequence (5 ' end vitamin H Biotin mark) of SEQ IDNO:5.
SEQ?ID?NO1：5’-CTTCCAgggCTCTCCCCTTgC-3’21bp
SEQ?ID?NO2：5’-AATTCTAATACgACTCACTATAggg?AAgATCAAAgAggTTCATg-3’44bp
SEQ?ID?NO3：5’HS-(CH
2)
6-[gAgCTgTAggAgTCTAAAT]19bp
SEQ?ID?NO4：5’[gACgTCACCTTgCAgTgTT]-(CH
2)
6-SH?3’19bp
SEQ?ID?NO5：5’BIOTIN-ggTCAACTCCATgATTAgCAT-3’21bp
Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';
Perhaps with above-mentioned sequence homology greater than 85% sequence;
The perhaps complementary base sequences thereof of above-mentioned sequence;
Perhaps use any one or more than one the combination in the above-mentioned all sequences.
In embodiments, the invention provides method and the test kit of a kind of augmentation detection HAV RNA based on the NITAG technology, described method comprises step:
(1) cooling after the RNA template is unwind under 60～70 ℃;
(2) add reversed transcriptive enzyme, RNase H and RNA polymerase, under the guiding of primer, carry out isothermal duplication, acquisition RNA amplicon in 37～42 ℃;
(3) utilize the oligonucleotide detection probes of nm gold particles mark and biotin labeled oligonucleotide capture probe to detect the RNA amplicon, obtain detected result by the color reaction in chromatographic film, the existence of colour developing band is the indication of hepatitis A virus (HAV) RNA in the sample.
The invention provides a kind of detection kit of utilizing above-mentioned detection method to carry out HAV RNA isothermal duplication, it consists of: the oligonucleotide capture probe of the oligonucleotide detection probes of NITAG reaction buffer, nm gold particles mark, vitamin H (or digoxin) mark and the chromatographic film that is fixed with avidin (or anti digoxin antibody) mark.The test kit that assembling forms is preserved down at-20 ℃.
Particularly, the present invention realizes in the following manner:
Hepatitis A virus (HAV) RNA NITAG detection technique of the present invention comprises that sample process, RNA isothermal duplication and Nano-Au probe detect three steps.The sample process step is to extract the process of RNA template from hepatitis A virus (HAV) cell culture sample, and augmentation detection is extracted the RNA template that obtains to liking from sample.A large amount of RNA amplicons that the hepatitis A virus (HAV) RNA template of utilizing extraction to obtain goes out through reversed transcriptive enzyme, ribonuclease H (RNase H), RNA polymerase (RNA Pol) isothermal duplication, and detect in conjunction with the Nano-Au probe of alkane sulfhydrylation oligonucleotide mark, realize detection by the chromatographic film color reaction to the RNA template.
Isothermal duplication system of the present invention comprises compositions such as RNA template, primer and damping fluid, detection architecture comprises compositions such as Nano-Au probe, capture probe and damping fluid, its concrete operations are: the RNA that sample to be measured is extracted inserts reaction buffer, ℃ following first aftertreatment 5min of (most preferably 65) ℃ and 37～42 (most preferably 41) 60～70, add mixed enzyme, (most preferably 41) ℃ following reaction 90～120min 37～42; And then the RNA amplified production mixed detection with nanometer gold detection probes and biotin labeling capture probe damping fluid, handle 5min cooling back chromatography developing on film at 37～65 ℃.
The final concentration of each component of isothermal amplification damping fluid of the present invention is: 20～60mM Tris-HCl (pH8.0～9.0), 30～90mM KCl, 10～20mM MgCl
2, 0.5～5mM deoxyribonucleoside triphosphate (dNTPs), 1～10mM ribonucleotide triphosphate (NTPs), 4～20mM dithiothreitol (DTT) (DTT), 10～20% (v/v) dimethyl sulfoxide (DMSO) (DMSO); Said two kinds of primers (comprising the primer I and the second chain cDNA synthetic primer II that have the sub-recognition sequence of rna polymerase promoter) final concentration respectively is: 0.1～1 μ mol/L; The amount of each component of said mixed enzyme solution in 20 μ L reaction systems is: 4～75U reversed transcriptive enzyme, 8～3000U RNA polymerase (the T3RNA polysaccharase or the t7 rna polymerase of identification specific promoter sequence), 0.05～0.3U RNase H enzyme, 10～50U RNA enzyme inhibitors (RNasin Inhibitor) and 0.05～5 μ g bSA (BSA).The final concentration of said each component of detection reaction damping fluid is: 0.3mol/L NaCl, 10mM phosphate buffered saline buffer (pH 7.0), 5 μ mol/L Nano-Au probes, 1 μ mol/L biotin labeling capture probe.Chromatographic film is nitrocellulose membrane or the nylon membrane (being stored in dry environment) that is fixed with avidin.
The used nm gold particles size of the present invention is 1～100nm, can adopt trisodium citrate reduction method, white phosphorus reduction method or xitix reduction method according to the particle diameter difference.
The preparation process of nanometer gold oligonucleotide detection probes of the present invention is:
(1) preparation of nm gold particles: boiling stirring HAuCl
4(1mM 500mL) under the situation of Hui Liuing, adds 50mL 38.8mM citric acid three sodium solution rapidly to solution, the color of solution will be become dark red by sallow, continue backflow 15min, be cooled to room temperature then, film with 0.45 μ M filters at last, promptly obtains the gold grain of about 13nm particle diameter.This nano-Au solution has the figuratrix plasma body absorption peak at 518～520nm place.Oligonucleotide probe length on the nm gold particles is 10～100bp, needs before the connection to handle and purifying through sulfhydrylation or alkane sulfhydrylation earlier.
The modification of the oligonucleotide of (2) 3 ' or 5 ' alkane marking sulfhydryls on nm gold particles: (it is synthetic to give birth to the worker by Shanghai to get nano-Au solution 5mL (about 17nm) 2.5OD alkyl oligonucleotide probe, dilution final concentration 3.61 μ mol/L) mix and hatch about 16 hours, then mixing solutions is placed 0.1mol/L NaCl, in the 10mM phosphate buffered saline buffer and kept 40 hours, next at the centrifugal at least 25min of 1400r/min to remove unreacted reagent.Remove supernatant liquor, remaining red oily precipitation is used buffer solution for cleaning again, is kept at 0.3mol/L NaCl after centrifugal, and in the 10mM phosphate buffered saline buffer (pH 7.0), the probe final concentration is 10 μ mol/L.Mensuration by infrared spectra proves that many probes have been modified on each gold grain surface, therefore just might form the converging network structure in the polynucleotide testing process.
(3) preparation of chromatographic film: the amount of 2mg/ml avidin with 2 μ l/cm is added on tunica fibrosa or the nylon membrane, and seasoning is 3 hours in the environment of 40 ℃ of humidity 40%, temperature; Then film is affixed on the fiber board that tears non-setting adhesive off, the upper end covers chromatographic film, compresses and stick non-setting adhesive and fix with filter paper, cuts into the wide test paper strip of 4mm standby (being stored in dry environment) with cutting the film machine at last.
Chromatographic film color reaction in the testing process of the present invention is a chromatography developing process behind the solution hybridization.The solution hybridization development process is about to isothermal duplication product 10 μ L (about 10pmol) and is added to and contains 20 μ L nanometer gold-alkane sulfhydrylation oligonucleotide detection probes, in the 1.5mL centrifuge tube of 10 μ L biotinylation capture probes, incubated at room 15min, then mixing solutions is placed constant temperature water bath and be heated 65 ℃, with mixture balance 5min, put into the chromatographic film test strip at last, through observing behind 5～10min, if Nano-Au probe and the hybridization of amplified production target sequence, can on film, form hepatic band, expression RNA detects positive, thereby show the existence of hepatitis A virus (HAV), otherwise will band can not occur.
Beneficial effect
The present invention has set up the method for utilizing NITAG technology for detection hepatitis A virus (HAV) or attenuated vaccine RNA, analyze from principle, isothermal duplication and Nano-Au probe be energy amplification detection signal all, what isothermal duplication amplified is the copy number of RNA molecule, what Nano-Au probe amplified is color signal, add that it is a narrow band that chromatographic step is assembled the hybridization complex of colour developing, the result observes more easy and directly perceived, so sensitiveer than the electrochemiluminescence detection technique after the NASBA amplification, make to be applied to HAV RNA and to detect and become possibility; Detect HAV relatively with fluorescent RT-PCR technology, its advantage is:
(1) because the NITAG technology can only the cloning RNA template, this can be avoided fluorescence RT-PCR DNA amplification product pollution to cause the generation of false positive detected result.
(2) only need conventional water-bath isothermal reaction just can realize detecting, need not expensive temperature cycling fluorescent PCR instrument.
(3) as seen detected result visual, very directly perceived by the colour developing band.
(4) because amplification efficiency is higher than the PCR reaction, and adopted the nano-gold signal amplification, detection sensitivity is higher.
(5) probe need not fluorescent mark, and it is lower that reagent detects cost.
Adopt the technology of the present invention, RNA amplification efficiency, detection sensitivity and the specificity of hepatitis A virus (HAV) or attenuated vaccine improve greatly, and have reduced the false positive rate that pcr amplification detects.Technical scheme of the present invention is reasonable in design, and the sample experiment can prove the existence of hepatitis A virus (HAV) or attenuated vaccine RNA on molecular level, show that this method is practical.Based on hepatitis A virus (HAV) or the attenuated vaccine RNA detection method and the test kit of NATAG technology, can be used as the assistant experiment means of estimating viral active state, have the clinical meaning and the using value of reality.
Description of drawings
Fig. 1 is a NITAG augmentation detection hepatitis A virus (HAV) RNA know-why synoptic diagram
Embodiment
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.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as the molecular cloning operational manual, or the condition of advising according to manufacturer.All inorganic chemical reagents and organic solvent are available from Shanghai chemical reagent factory; Trizol reagent is available from Invitrogen company; restriction enzyme is available from Shen, Shanghai energy lottery industry biotech firm; reverse transcription AMV enzyme, T7 RNA polymerase, RNase H, RNase Inhibitor and BSA are all available from U.S. Promega company, and primer and probe are given birth to worker company by Shanghai and synthesized.
The preparation and the composition of embodiment 1 hepatitis A virus (HAV) NITAG detection kit
Test kit is formed
(1) 2xNITAG reaction buffer
Each concentration of component is in per 10 μ l, 2 * NITAG reaction buffer: 80mM Tris-HCl (pH8.3), 160mM KCl, 24mM MgCl
2, 2mM dNTPs, 4mM NTPs, 10mM DTT, 30% (v/v) dimethyl sulfoxide (DMSO) (DMSO), two kinds of primers (SEQ ID No:1 and SEQ ID No:2) respectively are 0.4 μ mol/L.
(2) mixed enzyme
Contain 6.5U reversed transcriptive enzyme, 32U t7 rna polymerase, 0.1U RNaseH enzyme, 25U RNA enzyme inhibitors (RNasin Inhibitor) and 2.1 μ g bSAs (BSA) in per 5 μ l mixed enzyme.
(3) probe
Comprise the biotin labeled capture probes of 10 μ l, 4 μ mol/L (SEQID No:5), 20 μ l nm gold particles marker detection probes (each 10 μ l of SEQ ID No:3 and SEQ ID No:4).
(4) be fixed with the chromatographic film of avidin mark
The amount of 2mg/ml avidin with 2 μ l/cm is added on tunica fibrosa or the nylon membrane, and seasoning is 3 hours in the environment of 40 ℃ of humidity 40%, temperature; Then film is affixed on the fiber board that tears non-setting adhesive off, the upper end covers chromatographic film, compresses and stick non-setting adhesive and fix with filter paper, cuts into the wide test paper strip of 4mm standby (being stored in dry environment) with cutting the film machine at last
The test kit that assembling forms is preserved down at-20 ℃.
Provide reagent for oneself
Sterilization distilled water that chloroform, Virahol, DEPC handle and 75% ethanol of preparing with its.
The HAV RNA NITAG of embodiment 2 hepatitis A virus (HAV) cultures detects
Below detect and illustrate enforcement of the present invention by introducing RNA NITAG that hepatitis A virus (HAV) and hepatitis A virus (HAV) attenuated live vaccine extract.
RNA extracts: adopt hepatitis A to break out some a patient's ight soil or a serum sample, and hepatitis A virus (HAV) attenuated live vaccine sample, getting 250 μ l (ight soil get physiological saline rinsing after low-speed centrifugal supernatant liquor) respectively adds 500 μ l Trizol and extracts reagent, blow and beat mixing repeatedly with pipettor, room temperature was placed 5 minutes, add 200 μ l chloroforms, with vibrate back and forth mixing 15 seconds of hand, room temperature was placed 5 minutes, 4 ℃ 12, centrifugal 15 minutes of 000rpm, supernatant is moved into new centrifuge tube, add 200 μ l Virahols, vortex vibration 5 seconds, ice bath precipitation 10 minutes, 4 ℃ 12, centrifugal 10 minutes of 000rpm abandons supernatant, 75% washing with alcohol that adds the DEPC water preparation of 500 μ l precoolings, turn upside down 10 times, 4 ℃ 10, centrifugal 5 minutes of 000rpm, blot supernatant, precipitation drying at room temperature 10-20 minute adds the water dilution mixing that 10 μ l DEPC handle, and uses immediately and carries out the NITAG augmentation detection.
The NITAG augmentation detection: the RNA template after the extraction of getting 5 μ L is handled is inserted 10 μ L2x isothermal amplification system damping fluids, at 65 ℃ and 41 ℃ first aftertreatment 5min down, adds 5 μ L mixed enzyme solutions, reacts 120min down at 41 ℃; Getting 10 μ L isothermal duplication products is added in the 1.5mL centrifuge tube that contains 20 μ L nanometer gold-alkane sulfhydrylation oligonucleotide probe, 10 μ L biotinylation capture probes, incubated at room 15min, then mixing solutions is placed constant temperature water bath and be heated 65 ℃, with mixture balance 5min, put into the chromatographic film test strip at last, through observing behind 5～10min, form hepatic band on the chromatographic film, it is positive to illustrate that hepatitis A virus (HAV) or attenuated live vaccine RNA detect.
The nucleotides sequence tabulation
SEQUENCE?LISTING
＜110〉Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Fuxing Medical Science-Technology Development Co., Ltd., Shanghai
Wu Dazhi, Xia Yi
＜120〉a kind of method and test kit of nucleic acid isothermal augmentation detection hepatitis A virus (HAV)
<130>HAV?RNA
<140>CN
<141>2008-10-10
<160>5
<170>PatentIn?version?3.3
<210>1
<211>21
<212>DNA
<213>Hepatitis?A?virus
<400>1
cttccagggc?tctccccttg?c 21
<210>2
<211>44
<212>DNA
<213>Hepatitis?A?virus
<400>2
aattctaata?cgactcacta?tagggaagat?caaagaggtt?catg 44
<210>3
<211>20
<212>DNA
<213>Hepatitis?A?virus
<220>
＜221〉hexane sulfydryl
<222>(1)..(17)
<223>y＝HS-(CH2)6-
<400>3
ygagctgtag?gagtctaaat 20
<210>4
<211>20
<212>DNA
<213>Hepatitis?A?virus
<220>
＜221〉hexane sulfydryl
<222>(1)..(16)
<223>y＝-(CH2)6-SH
<400>4
gacgtcacct?tgcagtgtty 20
<210>5
<211>22
<212>DNA
<213>Hepatitis?A?virus
<220>
＜221〉vitamin H
<222>(1)..(19)
<223>y＝biotin-
<400>5
yggtcaactc?catgattagc?at 22
Claims (10)
1. a hepatitis A virus (HAV) (HAV) nucleic acid isothermal amplification detection method adopts reverse transcription-transcribe and Nano-Au probe detects the chromatography hybridization principle, its amplification object RNA template for extracting from HAV; It is characterized in that, the RNA template is by adopting reversed transcriptive enzyme, ribonuclease H and RNA polymerase isothermal duplication, the capture probe of a large amount of RNA amplicons that produce and the detection probes of nm gold particles mark, vitamin H (or digoxin) mark causes that in conjunction with forming hybridization complex optical property changes, at last this mixture and be fixed on avidin on the chromatographic film (or anti digoxin antibody) in conjunction with forming the color developing detection band.
2. detection method according to claim 1, adopted the oligonucleotide primer and the probe of amplification HAV target gene: primer has the sequence of SEQ ID NO:1 and SEQ ID NO:2, and wherein SEQ ID NO:25 ' end contains the promoter sequence of RNA polymerase identification; Detection probes has SEQ ID NO:3 and SEQ ID NO:4 sequence; Capture probe has SEQ I D NO:5 sequence.These primers and probe or include above-mentioned sequence to 5 ' end or and the sequence that prolongs of 3 ' end; Perhaps with above-mentioned sequence homology greater than 85% sequence; The perhaps complementary base sequences thereof of above-mentioned sequence; Perhaps use any one or more than one the combination in the above-mentioned all sequences.
3. detection method according to claim 1, its steps in sequence comprises:
(1) cooling after the RNA template is unwind under 60～70 ℃;
(2) add reversed transcriptive enzyme, ribonuclease H and RNA polymerase, under the guiding of primer, carry out isothermal duplication, acquisition RNA amplicon in 37～42 ℃;
(3) utilize the oligonucleotide probe of nm gold particles mark to detect amplicon, and caught, obtain detected result by the band color reaction on the chromatographic film by the combination of biotin labeling oligonucleotide probe.
4. detection method according to claim 1 is characterized in that, said reversed transcriptive enzyme is avian myeloblastosis virus reversed transcriptive enzyme or murine leukemia virus reverse transcriptase; Said RNA polymerase is T3RNA polysaccharase or t7 rna polymerase.
5. according to claim 1 and 5 said detection methods, it is characterized in that said T3RNA polysaccharase institute identification specificity promoter sequence is 5 ' AAT TCAATT AAC CCT CAC TAAAGGG3 '; Said t7 rna polymerase institute identification specificity promoter sequence is 5 ' AAT TCT AATACG ACT CAC TAT AGG G3 '.
6. detection method according to claim 1, it is characterized in that, used nm gold particles diameter is 1～100nm, and oligonucleotide probe SEQ ID NO:3 on the nm gold particles and SEQ ID NO:4 length are 10～100bp, and handles through sulfhydrylation or alkane sulfhydrylation before connecting; Used capture probe SEQ ID NO:5 length is 10～50bp, and its 5 ' end can be vitamin H or digoxigenin labeled.
7. detection method according to claim 1 is characterized in that, the used chromatographic film that is fixed with avidin or anti digoxin antibody (corresponding to capture probe 5 ' end mark vitamin H or digoxin) is nitrocellulose filter or nylon membrane.
8. detection method according to claim 1 is characterized in that, said color reaction bluish voilet colour developing band occurs for the hybridization back on chromatographic film.
9. test kit that utilizes the assembling of the described detection method of claim 1, it consists of: include oligonucleotide detection probes, vitamin H (or digoxin) mark of reaction buffer, the nm gold particles mark of primer I and II, reversed transcriptive enzyme, ribonuclease H, RNA polymerase oligonucleotide capture probe, have the chromatographic film of avidin (or anti digoxin antibody) mark.
10. a hepatitis A virus (HAV) nucleic acid isothermal amplification detection kit that utilizes the described detection method assembling of claim 1 can be used for the isolating RNA of hepatitis A virus (HAV) or attenuated hepatitis a vaccine and detect.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906473A (en) * | 2010-07-22 | 2010-12-08 | 中华人民共和国北京出入境检验检疫局 | Nucleic acid isothermal amplification kit and method for detecting EPSPS transgenic crops |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
| CN111690771A (en) * | 2020-06-08 | 2020-09-22 | 中联瑞(北京)生物科技有限责任公司 | Primer group, kit and method for detecting isothermal amplification of hepatitis A virus in food |
| CN114431560A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Mask for rapidly detecting new coronavirus and biological probe modification method |
-
2008
- 2008-10-24 CN CN200810201681A patent/CN101760562A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906473A (en) * | 2010-07-22 | 2010-12-08 | 中华人民共和国北京出入境检验检疫局 | Nucleic acid isothermal amplification kit and method for detecting EPSPS transgenic crops |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
| CN102154498B (en) * | 2011-03-21 | 2013-12-04 | 厦门大学 | Nucleic acid detecting method |
| CN111690771A (en) * | 2020-06-08 | 2020-09-22 | 中联瑞(北京)生物科技有限责任公司 | Primer group, kit and method for detecting isothermal amplification of hepatitis A virus in food |
| CN114431560A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Mask for rapidly detecting new coronavirus and biological probe modification method |
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