CN103382509A - Reagent and method for detecting dengue-2 virus through reverse transcription and loop-mediated isothermal amplification - Google Patents
Reagent and method for detecting dengue-2 virus through reverse transcription and loop-mediated isothermal amplification Download PDFInfo
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a reagent and a method for detecting dengue-2 virus through reverse transcription and loop-mediated isothermal amplification and can detect the dengue-2 virus efficiently and accurately. The reagent comprises an outer primer F3, an outer primer B3, an FIP (forward inner primer) and a BIP (backward inner primer). An RT-LAMP (reverse transcription-loop-mediated isothermal amplification) reaction system of the method is of 25 mu L and comprises primer mixture, template RNAs (ribonucleic acid), 8U Bst (Bacillus stearothermophilus) DNA (deoxyribonucleic acid) polymerases, 10U AMV (Avian Myelobastosis Virus) reverse transcriptase, glycine betaine, dNTP (deoxy-ribonucleoside triphosphate), MgSO4 and balance DEPC-H2O (Diethypyrocar-bonate water solution); an sample RNA, a positive control and a negative control are added into the system above and cultivated at a temperature of 63 DEG C for one hour for reaction; according to the electrophoretic results after the reaction, the positive control holes produce stepped strips, the negative control holes does not produce any strips, and if the detection sample holes produces stepped strips, dengue-2 virus exists, if not, dengue-2 virus does not exist.
Description
Technical field
The present invention relates to detect the detection method of reagent and the dengue 2-type virus of dengue 2-type virus, relate in particular to the method for utilizing reverse transcription-ring mediated isothermal amplification detection technique to detect dengue 2-type virus.
Background technology
Singapore hemorrhagic fever is the modal arthropod borne infections of the mankind, on the books since 16th century, is distributed widely in tropical and subtropical region, according to WHO, reports that the annual whole world approximately has new the infected of dengue virus of 50,000,000 to 100,000,000 to occur every year.Singapore hemorrhagic fever by dengue virus (dengue virus, DENV) cause, through her mosquito-borne acute infectious disease, dengue virus belongs to Alphaherpesvirinae (Togavirus) Flavivirus (Flavirus), comprises 1,2,3,4 four serotype.Clinical symptoms is that onset is anxious, heating, and whole-body muscle, marrow and arthrodynia, extremely tired, part is suffered from can fash, bleeding tendency and lymphadenectasis.The Category B notifiable disease that is defined as according to " People's Republic of China's law on the prevention and control of infectious diseases ".Since the end of the seventies, it is popular on a large scale that singapore hemorrhagic fever starts the whole world.Southeast Asian countries is the active area of dengue virus in nearly 30 years always, and since the nineties in last century, being very popular in various degree all occurs every year for South America and Caribbean.In recent years, along with the development of international trade, traffic, tourism, education etc., population mobile increasing, the Introduced cases singapore hemorrhagic fever happens occasionally.Research singapore hemorrhagic fever method for quick has outstanding practicality.Usually singapore hemorrhagic fever 2 C-type virus Cs are the most serious after infecting, dengue hemorrhagic fever easily occurs, the mortality ratio of dengue hemorrhagic fever reaches 15-50%, and the singapore hemorrhagic fever gene locus forms more complicated, universal singapore hemorrhagic fever test kit easily causes undetected, therefore studies the reagent that dengue 2-type virus detects.
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) is a kind of means of molecular Biological Detection fast, and characteristics are that reaction does not need temperature cycle, only need constant temp, can complete the amplification of target sequence.The method is 4 Auele Specific Primers of 6 zone design on target sequence, add the archaeal dna polymerase with strand displacement characteristic, do not need the DNA of thermally denature as template, can carry out nucleic acid amplification.Therefore this technology does not need special plant and instrument, and water-bath or incubator can meet the demands, and have higher application value, and the related personnel of grass-roots unit that can also be relatively poor as appointed condition carries out the instrument of conventional and Emergent detection.Add reversed transcriptive enzyme in reaction system after, can also from template ribonucleic acid, directly be detected by RT-LAMP (isothermal duplication of reverse transcription-ring mediation) single stage method.Reverse transcription and isothermal duplication a step at the same temperature complete, and have improved to a great extent the sensitivity detected and have saved the reaction times.
Summary of the invention
First purpose of the present invention is to provide the reagent that a kind of isothermal duplication detects dengue 2-type virus, for detecting efficiently and accurately dengue 2-type virus.For this reason, the present invention is by the following technical solutions:
It comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP; The primer gene order is as follows:
Outer primer F3:GAGCAGATCTCTGATGAATAACCAA, as shown in SEQ ID NO:2 in sequence table,
Outer primer R3:CATTCTGCCTGAGAAGAGGACAC, as shown in SEQ ID NO:3 in sequence table,
Inner primer FIP:
GAACAGTTTTAATGGTCCTCGTCCCGAGATTCTCACTTGGAATGCTGC, as shown in SEQ ID NO:4 in sequence table,
Inner primer BIP:
TGCTGAACATCTTGAACAGGAGACGCAACGCCATCACTGTTGGAATCA, as shown in SEQ ID NO:5 in sequence table.
Another object of the present invention is to provide a kind of method that constant-temperature amplification detects dengue 2-type virus, can detect rapidly and accurately dengue 2-type virus.For this reason, the present invention is by the following technical solutions:
The isothermal duplication RT-LAMP reaction system of reverse transcription-ring mediation is 25 μ L, and component is:
10×buffer2.5μL；
Template ribonucleic acid 2 μ L;
10U AMV reversed transcriptive enzyme 1 μ L;
The trimethyl-glycine 2 μ L of 8mol/L;
10mmol/L dNTP2μL；
100mmol/L MgSO
41.2μL；
Add DEPC-H
2o supplies 25 μ L;
Extract sample rna from 50 μ L sample serum, sample rna and positive control, negative control are added respectively in described RT-LAMP amplification system, negative control, positive control and detection sample are hatched to 63 ℃ of 1h by above-mentioned reaction system and reacted, wherein negative control adopts DEPC-H
2o, positive control adopts the outer transcribe rna of the non-infectosome of dengue 2-type virus; After reaction finishes, adopt at least one the method observations in following methods:
1), get amplified production and observe electrophoresis result, the positive control hole produces stair-stepping band, negative control hole does not have band to produce, and detects in sample aperture as produces stepped band to contain dengue 2-type virus, otherwise there is no dengue 2-type virus;
2), after reaction finishes, the centrifugal 6000rpm of reaction tubes, 3 minutes, observations, the visible white precipitate of positive control pipe, the negative control pipe does not produce precipitation, in sample tube as produce white precipitate and contain dengue 2-type virus, otherwise there is no dengue 2-type virus;
3), add 2 μ L fluorescence dye SYBR Green I or Eva Green in reaction system, observe the variation of fluorescence intensity under ultraviolet lamp, positive control produces strong green fluorescence, the negative control fluorescence intensity does not change, contain dengue 2-type virus as produced strong green fluorescence in sample tube, otherwise there is no dengue 2-type virus.
The contriver consults the dengue 2-type virus sequence at the GenBank database, dengue 2-type virus totally 73 strains of selecting the recent domestic different areas to find, with blast software, viral homologous sequence is compared, the position of target gene Piece Selection in dengue 2-type virus sequence gene group is between 95-627bp, totally 533 bases, the base sequence of described gene fragment is as shown in SEQ ID NO:1.4 primers of the present invention have been designed according to 6 specific regions in target gene.
The present invention has advantages of the following aspects: (1) design of primers is at the conservative region of dengue 2-type virus genome sequence, with the GenBank database, retrieved, without intersection, guaranteed the specificity of detection method with the nucleotide sequence of other subtypes of dengue virus and other species.(2) highly sensitive, the lowest detection of reaction is limited to the 2pg left and right.(3) amplification efficiency is high, and the reaction times is short, and the detection of sample only needs can complete about 1 hour.(4) this detection method is without expensive plant and instrument, and the rapid screening that is applicable to the basic unit port detects.
The accompanying drawing explanation
Fig. 1 a is the specific test electrophoresis result of stepping on leather 2 viral isothermal duplications in embodiment 1, wherein M is marker, 1 positive contrast, 2 is dengue 2-type virus, 3 is dengue 1-type virus, 4 is dengue 3 virus, 5 is 4-type dengue virus, and 6 is datum hole Kenya virus, and 7 is the yellow fever virus vaccine, 8 is encephalitis b virus, 9 negative contrasts.
Fig. 1 b is the specific test centrifugation result of stepping on leather 2 viral isothermal duplications in embodiment 1, wherein 1 positive contrast, 2 is dengue 2-type virus, 3 is dengue 1-type virus, and 4 is dengue 3 virus, and 5 is 4-type dengue virus, 6 is datum hole Kenya virus, 7 is the yellow fever virus vaccine, and 8 is encephalitis b virus, 9 negative contrasts.
Fig. 1 c is the specific test fluorescent reaction result of stepping on leather 2 viral isothermal duplications in embodiment 1, wherein 1 positive contrast, 2 is dengue 2-type virus, 3 is dengue 1-type virus, and 4 is dengue 3 virus, and 5 is 4-type dengue virus, 6 is datum hole Kenya virus, 7 is the yellow fever virus vaccine, and 8 is encephalitis b virus, 9 negative contrasts.
Fig. 2 is the result after the different magnesium ion concentration amplifications of reaction system in embodiment 1, and wherein M is marker, and 1 is 100mmol/L MgSO
41.0 μ L, 2 is 100mmol/L MgSO
41.1 μ L, 3 is 100mmol/LMgSO
41.2 μ L, 4 is 100mmol/L MgSO
41.3 μ L, 5 negative contrasts.
Fig. 3 is the electrophoresis result increased at different temperature in embodiment 1, and wherein M is marker, and 1 is 61 ℃, and 2 are 62 ℃, 3 is 63 ℃, 4 to be 64 ℃, 5 be 65 ℃.
The sensitivity test result that Fig. 4 is dengue 2-type virus isothermal duplication in embodiment 1, wherein M is marker, and 1 is 200ng, and 2 is 20ng, and 3 is 2ng, and 4 is 200pg, and 5 is 20pg, and 6 is 2pg, 7 is 0.2pg, 8 negative contrasts.
Embodiment
The detection of embodiment 1 dengue 2-type virus
1. design of primers
Consult the dengue 2-type virus sequence at the GenBank database, dengue 2-type virus totally 73 strains of selecting the recent domestic different areas to find, compare to viral homologous sequence with blast software, according to 4 primers of 6 specific regions designs in target gene,
Outer primer F3:GAGCAGATCTCTGATGAATAACCAA;
Outer primer R3:CATTCTGCCTGAGAAGAGGACAC;
Inner primer FIP:
GAACAGTTTTAATGGTCCTCGTCCCGAGATTCTCACTTGGAATGCTGC；
Inner primer BIP:
TGCTGAACATCTTGAACAGGAGACGCAACGCCATCACTGTTGGAATC A。
2. the extraction of dengue 2-type virus RNA
To require to extract dengue 2-type virus RNA ,-70 ℃ of preservations according to QIAGEN nucleic acid extraction kit specification sheets.
3.RT-LAMP reaction system and reaction conditions
The isothermal duplication RT-LAMP reaction system of reverse transcription-ring mediation is 25 μ L, and component is:
10×buffer2.5μL；
Template ribonucleic acid 2 μ L;
10U AMV reversed transcriptive enzyme 1 μ L;
The trimethyl-glycine 2 μ L of 8mol/L;
10mmol/L dNTP2μL；
Regulate MgSO
4(100mmol/L) consumption;
Add DEPC-H
2o supplies 25 μ L; Between temperature of reaction is selected 61-65 ℃, reaction 60min, according to the reaction conditions of the selection optimum of amplification efficiency.
4. reaction system Mg
2+the optimization of concentration
The required best Mg of research reaction system
2+concentration is regulated MgSO in the reaction system of aforementioned the 3rd
4consumption, add respectively MgSO
4(100mmol/L) 1.0 μ L, 1.1 μ L, 1.2 μ L, 1.3 μ L, add DEPC-H
2o supplies 25 μ L.With DEPC-H
2the negative contrast of O, 63 ℃, 60 minutes.Reaction result, as Fig. 3, in 25 μ L systems, adds the MgSO that concentration is 100mmol/L
41.2 μ L(Mg
2+final concentration is 4.8mmol/L) time amplification efficiency for the highest.
5. the optimization of temperature of reaction
For selecting the highest temperature of reaction of amplification efficiency, adopt respectively 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃ to be stepped on leather 2 type RT-LAMP amplified reactions, according to above-mentioned best Mg
2+the preparation reaction system of concentration, the RT-LAMP reaction system is as follows: 10 * buffer2.5 μ L, in primer mixture 1 μ L(system, the primer final concentration is as follows respectively: each 40pmol/L of F3, B3, each 800pmol/L of FIP, BIP), template ribonucleic acid 2 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, add DEPC-H
2o supplies 25 μ L, with DEPC-H
2the negative contrast of O, 60 minutes reaction times.The relatively impact of differing temps on reaction, reaction result is shown in Fig. 2, between 61-65 ℃, 63 ℃ is best reaction amplification temperature.
6. specific test
In order to verify the specificity of detection reagent of the present invention and method, by 4 kinds of hypotypes of dengue virus and yellow fever virus and datum hole Kenya virus, encephalitis b virus adopts detection reagent of the present invention and method to detect simultaneously, dengue virus is arboviruses flaviviridae Flavivirus, by the yellow fever virus vaccine, Chikungunya virus, the flavivirus such as encephalitis b virus extract RNA with RNA extraction agent box according to specification sheets, adopt dengue 2-type virus RT-LAMP method to be detected, the RT-LAMP reaction system is as follows: 10 * buffer2.5 μ L, in primer mixture 1 μ L(system, the primer final concentration is as follows respectively: F3, each 40pmol/L of B3, FIP, each 800pmol/L of BIP), template ribonucleic acid 2 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, add DEPC-H
2o supplies 25 μ L.The reaction electrophoresis result is shown in Fig. 1 a, the centrifugal 6000rpm of reaction tubes, after 3 minutes, the results are shown in Figure 1b, adds 2 μ L fluorescence dye SYBR Green I, observes the 1c that the results are shown in Figure of fluorescence intensity under ultraviolet lamp.
Reaction result shows to occur the positive findings of typical stepped band except singapore hemorrhagic fever 2 viral nucleic acids, the detected result of negative control, dengue 1-type virus, dengue 3 virus, 4-type dengue virus, yellow fever virus vaccine, datum hole Kenya virus, encephalitis b virus is all negative, has shown that the method has good specificity.
7. sensitivity test
By the dengue 2-type virus RNA of aforementioned extraction, measuring by Eppendorf nucleic acid-protein determinator the viral RNA concentration of extracting is 100ng/ μ l.The dengue 2-type virus geneome RNA is carried out to 10 times of dilutions of going forward one by one, and (concentration is respectively: 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg), getting respectively 2 μ l adds in RT-LAMP reaction system separately as template, the RT-LAMP reaction system is as follows: 10 * buffer2.5 μ L, in primer mixture 1 μ L(system, the primer final concentration is as follows respectively: F3, each 40pmol/L of B3, FIP, each 800pmol/L of BIP), template ribonucleic acid 2 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, add DEPC-H
2o supplies 25 μ L.63 ℃, 60 minutes.Get respectively 2 μ l reaction product and carry out 1% agarose gel electrophoresis, reaction result is shown in Fig. 4, and stepped band all appears in the 2-7 swimming lane, shows to have positive RT-LAMP amplified reaction.The limit of detection that can judge present method is about 2fg.
Adopt dengue 2-type virus sample of nucleic acid 3 examples, port, Zhejiang Province immigration heating personnel 21 routine serum, utilize RNA extraction agent box to extract nucleic acid according to specification sheets, get respectively 2 μ l and add RT-LAMP reaction system separately, the RT-LAMP reaction system is as follows: 10 * buffer2.5 μ L, in primer mixture 1 μ L(system, the primer final concentration is as follows respectively: F3, each 40pmol/L of B3, FIP, each 800pmol/L of BIP), template ribonucleic acid 2 μ L, 8U BstDNA polysaccharase 1 μ L, 10U AMV reversed transcriptive enzyme 1 μ L, trimethyl-glycine (8mol/L) 2 μ L, dNTP (10mmol/L) 2 μ L, MgSO
4(100mmol/L) 1.2 μ L, add DEPC-H
2o supplies 25 μ L.With DEPC-H
2the negative contrast of O, with the outer positive contrast of transcribe rna of the non-infectosome of dengue 2-type virus.Response procedures is 63 ℃, 60 minutes.Centrifugal 6000rpm after 3 routine dengue 2-type virus sample reactions finish, after 3 minutes, visible white precipitate, add 2 μ L fluorescence dye SYBR Green I, under ultraviolet lamp, has strong green fluorescence to produce, special stepped band appears in the product electrophoresis, and the dengue 2-type virus detected result is positive.The centrifugal 6000rpm of 21 routine patient-heated serum's sample reaction result, within 3 minutes, have no precipitation, add under 2 μ L fluorescence dye SYBR Green I ultraviolet lamps and do not have green fluorescence to produce, special stepped band does not appear in the product electrophoresis, and the dengue 2-type virus detected result is negative.
<110 > Zhejiang international travel health care center
<120 > reverse transcription-ring mediated isothermal amplification detects reagent and the detection method of dengue 2-type virus
<130>
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<170> PatentIn version 3.3
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gagcagatct ctgatgaata accaacgaaa aaaggcgaga aatacgcctt tcaatatgct 60
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aatgctgcag ggacgaggac cattaaaact gttcatggcc ctggtggcgt tccttcgttt 180
cctaacaatc ccaccaacag cagggatact gaagagatgg ggaacaatta aaaaatcaaa 240
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caggagacgc agaactgcag gcatgatcat tatgctgatt ccaacagtga tggcgttcca 360
tttaaccaca cgtaacggag aaccacacat gatcgtcagt agacaagaga aagggaaaag 420
tcttctgttt aaaacagggg atggtgtgaa catgtgtacc ctcatggcca tggaccttgg 480
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Claims (2)
1. a reverse transcription-ring mediated isothermal amplification detects the reagent of dengue 2-type virus, and it is characterized in that: it comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP; The primer gene order is as follows:
Outer primer F3:GAGCAGATCTCTGATGAATAACCAA;
Outer primer R3:CATTCTGCCTGAGAAGAGGACAC;
Inner primer FIP:
GAACAGTTTTAATGGTCCTCGTCCCGAGATTCTCACTTGGAATGCTGC；
Inner primer BIP:
TGCTGAACATCTTGAACAGGAGACGCAACGCCATCACTGTTGGAATCA。
2. a reverse transcription-ring mediated isothermal amplification detects the method for dengue 2-type virus, it is characterized in that:
The isothermal duplication RT-LAMP reaction system of reverse transcription-ring mediation is 25 μ L, and component is:
10×buffer2.5μL；
Primer mixture 1 μ L, inner primer FIP claimed in claim 1 and the concentration of inner primer BIP in described reaction system are 800pmol/L, and outer primer F3 and the concentration of outer primer B3 concentration in described reaction system are 40pmol/L;
Template ribonucleic acid 2 μ L;
8U BstDNA polysaccharase 1 μ L;
10U AMV reversed transcriptive enzyme 1 μ L
The trimethyl-glycine 2 μ L of 8mol/L;
10mmol/L dNTP2μL；
100mmol/L MgSO
41.2μL；
Add DEPC-H
2o supplies 25 μ L;
Extract sample rna, sample rna and positive control, negative control are added respectively in described RT-LAMP amplification system, negative control, positive control and detection sample are hatched to 63 ℃ of 1h by above-mentioned reaction system and reacted, wherein negative control adopts DEPC-H
2o, positive control adopts the outer transcribe rna of the non-infectosome of dengue 2-type virus; After reaction finishes, adopt at least one the method observations in following methods:
1), get amplified production and observe electrophoresis result, the positive control hole produces stair-stepping band, negative control hole does not have band to produce, and detects in sample aperture as produces stepped band to contain dengue 2-type virus, otherwise there is no dengue 2-type virus;
2), after reaction finishes, the centrifugal 6000rpm of reaction tubes, 3 minutes, observations, the visible white precipitate of positive control pipe, the negative control pipe does not produce precipitation, in sample tube as produce white precipitate and contain dengue 2-type virus, otherwise there is no dengue 2-type virus;
3), add 2 μ L fluorescence dye SYBR Green I or Eva Green in reaction system, observe the variation of fluorescence intensity under ultraviolet lamp, positive control produces strong green fluorescence, the negative control fluorescence intensity does not change, contain dengue 2-type virus as produced strong green fluorescence in sample tube, otherwise there is no dengue 2-type virus.
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| CN110894552A (en) * | 2019-11-08 | 2020-03-20 | 珠海国际旅行卫生保健中心(拱北海关口岸门诊部) | Primers, Probes, Kits and RT-iiPCR Methods for Dengue Virus Detection |
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