CN104946792A - RT-LAMP kit for rapidly identifying dengue virus type 4 - Google Patents
RT-LAMP kit for rapidly identifying dengue virus type 4 Download PDFInfo
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Abstract
The present invention discloses a RT-LAMP kit for rapidly identifying dengue virus type 4. The kit comprises a pair of outer primers, a pair of inner primer and a pair of ring primers, wherein the sequences are respectively represented by SEQ ID NO.1-6. The kit further comprises a primer solution, a reaction solution, DNA polymerase, reverse transcriptase, controls and a chromogenic agent. The detection method comprises: extracting RNA of virus to be detected, using reverse transcription activity of reverse transcriptase, using six specific primers and DNA polymerase having strand displacement activity, amplifying the sample RNA template at a temperature of 60-65 DEG C, and adding a chromogenic agent to observe whether the color in the reaction tube is changed so as to determine the amplification. The kit of the present invention has characteristics of rapidness, high efficiency, easy operation, high specificity, high sensitivity, easy identification, field detection, and the like, and is suitable for promotion application.
Description
Technical field
The present invention relates to the Detection and Identification of pathogenic micro-organism, be specifically related to the method for one ring mediated isothermal gene amplification (LAMP technology) Rapid identification 4 type dengue fever virus.
Background technology
Dengue fever virus (Dengue virus, DENV) is a kind of flavivirus RNA viruses, mainly comprises 1 ~ 4 C-type virus C (DENV 1 ~ 4).Dengue fever virus mainly through Aedes aegypti (
aedes aegypti) and Aedes albopictus (
aedes albopictus) propagate, the infection of dengue fever virus can cause singapore hemorrhagic fever (dengue fever, DF), and serious also can cause dengue hemorrhagic fever (Dengue hemorrhagic fever, or dengue shock syndrome (Dengue shocksyndrome, DSS) etc. DHF).DENV is widely current in the subtropical and tropical zones in the whole world, and China is mainly distributed in Guangdong, Hainan, Guangxi, Fujian, Taiwan etc. and economizes (autonomous region).ENV-4 type spreads along West Coast to north and south for 1979 from northern Baima town, Danxian County, Hainan Island, involves the North Sea in areas such as bearing river, Foshan, Guangzhou, Shantou, Shaoguan and Guangxi, Hepu, popular 3 years continuously.
The authentication method of current dengue fever virus is mainly PCR, the method such as ELISA, immune colloid gold.Singapore hemorrhagic fever detects conventional ELISA, immune colloidal gold method length consuming time, just can reach antibody in usual needs 5 ~ 7 d serum can detection level, there is cross reaction in dengue antibody and other flaviviruss simultaneously, occur that false positive rate is higher, therefore Serologic detection is larger as dengue fever virus detection limitation; The separation of DENV can well identify dengue fever virus, but same length consuming time, susceptibility is very low again, also cannot as the means of early detection dengue fever virus.The DENV nucleic acid detection technique that nearly 10 years development are got up, for the early diagnosis of singapore hemorrhagic fever provides possibility, but PCR and RT-PCR needs based on expensive PCR instrument, also limits the development of PCR method on qualification dengue fever virus to a certain extent.
Although various method may provide good value clinically above, because length consuming time, loaded down with trivial details operating process, sensitivity are low, need the different reasons such as expensive plant and instrument to fail large-scale promotion clinically to use.Ring mediated isothermal gene amplification (Loop-Mediated Isothermal Amplification, hereinafter referred to as LAMP method) be the gene amplification that Japanese Eiken Chemical developed before and after 2000, it has fast and convenient, that operation is accurate, easily universal, safe and reliable advantage, not yet has at present and LAMP method is applied to Rapid identification 4 type dengue fever virus.
Summary of the invention
The object of the present invention is to provide a kind of method of RT-LAMP test kit for Rapid identification 3 type dengue fever virus and one ring mediated isothermal gene amplification (LAMP technology) Rapid identification 4 type dengue fever virus.This authentication method is convenient and swift, cheap, is suitable for applying.
The technical solution used in the present invention is:
For a RT-LAMP test kit for Rapid identification 4 type dengue fever virus, comprise following component: (1) is for the RT-LAMP Auele Specific Primer group of Rapid identification 4 type dengue fever virus; (2) reversed transcriptive enzyme; (3) archaeal dna polymerase; (4) RT-LAMP reaction solution; (5) developer; (6) positive control and negative control; Wherein, the described RT-LAMP Auele Specific Primer group for Rapid identification 4 type dengue fever virus comprises:
Inner primer 1:5 '-CGTTATTGGCGGAGCTACAGGGAGGCTATTGAAGTCAGGC-3 ' (SEQ ID No.1);
Inner primer 2:5 '
-GTGGACTAGCGGTTAGAGGAGAGAGACTACAGCTTCATCTCAC-3’ （SEQ ID No.2）；
Outer primer 1:5 '-GCTCCTTTCGAGAGTGAAG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-GGTCTCCTCTAACCTCTAGTC-3 ' (SEQ ID No.4);
Ring primer 1:5 '-CAGCACGGTTTGCTCAAG-3 ' (SEQ ID No.5);
Ring primer 2: 5 '-CCTTACAAATCGCAGCAACAA-3 ' (SEQ ID No.6).
In described primer sets, the mol ratio of inner primer, outer primer, ring primer is 6 ~ 8:1:3 ~ 4.
Described reversed transcriptive enzyme is
aMVreversed transcriptive enzyme; Described archaeal dna polymerase is
bstarchaeal dna polymerase.
Described RT-LAMP reaction solution contains: 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM MgSO
4solution, 5mM trimethyl-glycine, the volume ratio of four is 6 ~ 8:4 ~ 5:2 ~ 3:8 ~ 10.
Described developer is SYBR GREEN I.
Described positive control is the plasmid containing 4 type dengue fever virus NS1 gene fragments (SEQ ID NO.7), and negative control is DEPC water.
A method for Rapid identification dengue fever virus 4 type, comprise and adopt above-described test kit, add template ribonucleic acid, at 60 ~ 65 DEG C, isothermal duplication 60 ~ 90 minutes, specifically comprises the steps:
(1) template ribonucleic acid is extracted;
(2) ring mediated isothermal gene amplification reaction: 25 μ l reaction systems contain: each 7 ~ 9pmol/ μ l of inner primer 1,2, each 1 ~ 2 pmol/ μ l of outer primer 1,2, each 3 ~ 5 pmol/ μ l of ring primer 1,2, reaction solution 12 ~ 13 μ l, archaeal dna polymerase 7 ~ 9U, RNA 1 ~ 100ng to be checked, reversed transcriptive enzyme 1 ~ 2U, with sterilizing deionized water polishing to 25 μ l, then add the sealing liquid of 18 ~ 22 μ l, by above-mentioned reaction tubes in 60 ~ 65 DEG C of reaction 60 ~ 90min;
(3) result judges: in above-mentioned reaction tubes, add 1-2 μ l developer 10 × SYBR GREEN I, and the color of observing response liquid after mixing, if present green, be singapore hemorrhagic fever 4 C-type virus C, orange is then non-singapore hemorrhagic fever 4 C-type virus C.
Preferably, 25 μ l reaction systems of described ring mediated isothermal gene amplification reaction contain: each 8pmol/ μ l of inner primer 1,2, the each 1 pmol/ μ l of outer primer 1,2, the each 4 pmol/ μ l of ring primer 1,2, reaction solution 12.5 μ l, archaeal dna polymerase 8U, RNA 1 ~ 100ng to be checked, reversed transcriptive enzyme 1U, with sterilizing deionized water polishing to 25 μ l.
The invention has the beneficial effects as follows: the present invention is directed to 4 type dengue fever virus NS1 regiospecificities and devise 6 primers, be combined with 6 regions of goal gene, there is higher specificity.Test kit of the present invention is convenient and swift, can differentiate 4 type dengue fever viruss in the short period of time, and does not need expensive plant and instrument; Highly sensitive; Specificity is good, is applicable to Site Detection.
Accompanying drawing explanation
Fig. 1 be the feminine gender of RT-LAMP test kit of the present invention and positive reference substance detected result (-: negative control ,+: positive control);
Fig. 2 be the present invention to singapore hemorrhagic fever 4 C-type virus C and to the amplification of non-singapore hemorrhagic fever 4 C-type virus C (-: negative sample ,+: positive, 1: non-4 type dengue fever virus samples, 2:4 type dengue fever virus sample);
Fig. 3 is the specificity verification result (1:DEPC, 2:HSV, 3:EBV, 4:JEV, 5:YFV, 6:DENV1,7:DENV2,8:DENV3,9:DENV4) of embodiment 3;
Fig. 4 is the sensitivity experiment result (a: regular-PCR amplification of embodiment 4, b:Real-time pcr amplification result, c: the electrophoresis result of RT-LAMP amplification of the present invention, d: the Real-time result of RT-LAMP amplification of the present invention, e: the colour developing result of RT-LAMP amplification of the present invention; Wherein, 1 is DEPC, and 2 ~ 7 is the PET-32a plasmid containing dengue fever virus UTR gene, and copy number is followed successively by 1.0,1.0 × 10
1, 1.0 × 10
2, 1.0 × 10
3, 1.0 × 10
4, 1.0 × 10
5copy/μ L).
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1 is for the foundation of the RT-LAMP test kit of Rapid identification 4 type dengue fever virus
For the RT-LAMP test kit of Rapid identification 4 type dengue fever virus, comprise following composition: (1) Auele Specific Primer group; (2) reversed transcriptive enzyme; (3) archaeal dna polymerase; (4) RT-LAMP reaction solution; (5) developer; (6) positive control and negative control.
(1) design of Auele Specific Primer:
According to 4 type dengue fever viruss (GenBank accession number is: Y19176) UTR region specific designs 6 Auele Specific Primers, sequence is respectively as follows:
Inner primer 1:5 '-CGTTATTGGCGGAGCTACAGGGAGGCTATTGAAGTCAGGC-3 ' (SEQ ID No.1);
Inner primer 2:5 '
-GTGGACTAGCGGTTAGAGGAGAGAGACTACAGCTTCATCTCAC-3’（SEQ ID No.2）；
Outer primer 1:5 '-GCTCCTTTCGAGAGTGAAG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-GGTCTCCTCTAACCTCTAGTC-3 ' (SEQ ID No.4).
Ring primer 1:5 '-CAGCACGGTTTGCTCAAG-3 ' (SEQ ID No.5)
Ring primer 2: 5 '-CCTTACAAATCGCAGCAACAA-3 ' (SEQ ID No.6)
(2) reversed transcriptive enzyme is
aMVreversed transcriptive enzyme;
(3) archaeal dna polymerase is
bstarchaeal dna polymerase;
(4) RT-LAMP reaction solution contains: 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM MgSO
4, 5mM trimethyl-glycine, the volume ratio of four is 8:5:3:10.
(5) developer is SYBR GREEN I.
(6) positive control is the PET-32a plasmid (utilizing conventional plasmid construction method to be inserted in PET-32a plasmid by SEQ ID NO.7 to obtain) containing 4 type dengue fever virus UTR gene fragments (SEQ ID NO.7); Negative control is DEPC water.
The amplification of positive control and negative control is shown in Fig. 1.
embodiment 2 utilizes the test kit Rapid identification 4 type dengue fever virus of embodiment 1
Concrete steps are as follows:
(1) template ribonucleic acid is extracted: adopt viral RNA to extract test kit and extract sample RNA;
(2) ring mediated isothermal gene amplification reaction: 25 μ l reaction systems contain: inner primer 1,2 final concentration is respectively 8pmol/ μ l, outer primer 1,2 final concentration is respectively 1 pmol/ μ l, ring primer 1,2 final concentration is respectively 4 pmol/ μ l, reaction solution 12.5 μ l, archaeal dna polymerase 8U, RNA 50ng to be checked, reversed transcriptive enzyme 1U, with sterilizing deionized water polishing to 25 μ l, then add the sealing liquid of 20 μ l, by above-mentioned reaction tubes in 65 DEG C of reaction 60 min;
(3) result judges: in above-mentioned reaction tubes, add 1-2 μ l developer 1 × SYBR Green I, and the color of observing response liquid after mixing, if present green, be singapore hemorrhagic fever 4 C-type virus C, orange is then non-singapore hemorrhagic fever 4 C-type virus C (as Fig. 2).
embodiment 3 specificity experiments
Utilize the method for embodiment 2 to detect 2 kinds of spore exanthemas, encephalitis b virus, yellow fever virus, 1 type dengue fever virus, 2 type dengue fever viruss, 3 type dengue fever viruss, 4 type dengue fever viruss respectively, DEPC water belongs with yin contrasts.
Detected result is shown in Fig. 3.Only 4 type dengue fever virus pipes are green, and all the other pipes are for orange.Result shows, detection kit specificity of the present invention is high, can 4 type dengue fever viruss and 1,2,3 type dengue fever viruss and other irrelevant virus be made a distinction (Fig. 3) exactly.
embodiment 4 sensitivity experiment
Get positive reference substance (namely build containing the PET-32a plasmid of 4 type dengue fever virus UTR gene fragments (SEQ ID NO.7)), measure its concentration and calculate the copy number of NS4A gene, according to the concentration gradients dilution of 10 times, choosing 1.0 × 10
0~ 1.0 × 10
5the concentration of copy/μ L is tested as sample.Identify by authentication method of the present invention and conventional PCR method and Real-time PCR method respectively.
1, LAMP-PCR detection method of the present invention
The positive reference substance of each concentration respectively gets 1 μ L, adopts the method for embodiment 2 to detect respectively, and experimental result is shown in c, d, e:1.0 in Fig. 4 × 10
5, 1.0 × 10
4, 1.0 × 10
3, 1.0 × 10
2, 1.0 × 10
1the PCR pipe of individual copy is green, indicates amplified reaction; 1.0 copy and negative control PCR pipe be orange, show without amplified reaction.LAMP shows experimental result by 3 kinds of methods such as colour developing, electrophoresis, fluorescence curves, all occurs consistent result.
2, Standard PCR detection method
(1) PCR primer:
F： GCTCCTTTCGAGAGTGAAG（SEQ ID NO.8），
R： GGTCTCCTCTAACCTCTAGTC（SEQ ID NO.9）。
(2) PCR reaction: PCR reaction system is 25 μ L systems, 10 × PCR Buffer(PCR reaction buffer, Promega company) 2.5 μ L, 10mM dNTPs (Promega company) 0.5 μ L, the final concentration of upstream and downstream primer is respectively 0.4 μm of ol/L, Taq enzyme (5U/ μ L, Promega company) 0.5 μ L, the positive reference substance of above-mentioned each concentration respectively gets 1 μ L as pcr template, mends to 25 μ L with sterilizing deionized water.Response procedures is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, and 72 DEG C extend 30s, and 72 DEG C extend 7min.PCR primer gets 10 μ L and 2% agarose gel electrophoresis, 30min under 100V voltage, and by gel image analyser observations, expection object band is 385bp.Result display 1.0 × 10
1, 1.0 × 10
0the PCR pipe of individual copy is shown as feminine gender, namely without amplification (see in Fig. 4 a).
3, Real-time PCR detects
(1) Real-time PCR primer:
F：GCTCCTTTCGAGAGTGAAG（SEQ ID NO.8），
R：GGTCTCCTCTAACCTCTAGTC（SEQ ID NO.9）。
(2) Real-time PCR reacts: reaction system is 25 μ L systems, 2 × SYBR Green II buffer 5 μ L, the final concentration of upstream and downstream primer is respectively 0.4 μm of ol/L, and the positive reference substance of above-mentioned each concentration respectively gets 1 μ L as pcr template, mends to 25 μ L with sterilizing deionized water.Response procedures is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 55 DEG C of annealing 20s, and 72 DEG C extend 20s, and 72 DEG C extend 7min.By fluorescence curve judgment experiment result.Experimental result is shown in the b in Fig. 4, result display 1.0 × 10
1, 1.0 × 10
0not there is amplified peak in the PCR pipe of individual copy, namely without amplification.
embodiment 5
The method of embodiment 2 is utilized to carry out process inspection to multiple dengue fever virus 3 type disease human blood sample that has been defined as, process multiple patient's sample, wherein only have a patient to detect result for negative, all the other patients are the positive (table 1), and accuracy can reach more than 95%.
table 1 the present invention is to the inspection of clinical dengue fever virus 4 type patient
Note: "+" represents that detected result is positive, namely has amplification; "-" represents that detected result is negative, namely without amplification.
Above embodiment shows, test kit of the present invention has good, the highly sensitive feature of accuracy, and does not need expensive plant and instrument, is applicable to Site Detection.
Above embodiment is only introduces preferred case of the present invention, to those skilled in the art, not deviating from any apparent changes and improvements of carrying out in the scope of spirit of the present invention, all should be regarded as a part of the present invention.
<110> Zhongshan University
<120> mono-kind is for the RT-LAMP test kit of Rapid identification 4 type dengue fever virus
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aatttttgag gctattgaag tcaggccact tgtgccacgg cttgagcaaa ccgtgctgcc 120
tgtagctccg ccaataacgg gaggcgttaa attcccaggg aggccatgcg ccacggaagc 180
tgtacgcgtg gcatattgga ctagcggtta gaggagaccc ctcccatcac taacaaaacg 240
cagcaaaagg ggggcccgaa gccaggagga agctgtactc ctgg 284
<210> 8
<211> 19
<212> DNA
<213> artificial sequence
<400> 8
gctcctttcg agagtgaag 19
<210> 9
<211> 21
<212> DNA
<213> artificial sequence
<400> 9
ggtctcctct aacctctagt c 21
Claims (8)
1. for a RT-LAMP test kit for Rapid identification 4 type dengue fever virus, comprise following component: (1) is for the RT-LAMP Auele Specific Primer group of Rapid identification 4 type dengue fever virus; (2) reversed transcriptive enzyme; (3) archaeal dna polymerase; (4) RT-LAMP reaction solution; (5) developer; (6) positive control and negative control; Wherein, the described RT-LAMP Auele Specific Primer group for Rapid identification 4 type dengue fever virus comprises:
Inner primer 1:5 '-CGTTATTGGCGGAGCTACAGGGAGGCTATTGAAGTCAGGC-3 ' (SEQ ID No.1);
Inner primer 2:5 '
-GTGGACTAGCGGTTAGAGGAGAGAGACTACAGCTTCATCTCAC-3’ （SEQ ID No.2）；
Outer primer 1:5 '-GCTCCTTTCGAGAGTGAAG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-GGTCTCCTCTAACCTCTAGTC-3 ' (SEQ ID No.4);
Ring primer 1:5 '-CAGCACGGTTTGCTCAAG-3 ' (SEQ ID No.5);
Ring primer 2: 5 '-CCTTACAAATCGCAGCAACAA-3 ' (SEQ ID No.6).
2. the RT-LAMP test kit for Rapid identification 4 type dengue fever virus according to claim 1, is characterized in that, in described primer sets, the mol ratio of inner primer, outer primer, ring primer is 6 ~ 8:1:3 ~ 4.
3. the RT-LAMP test kit for Rapid identification 4 type dengue fever virus according to claim 1, it is characterized in that, described reversed transcriptive enzyme is
aMVreversed transcriptive enzyme; Described archaeal dna polymerase is
bstarchaeal dna polymerase.
4. the RT-LAMP test kit for Rapid identification 4 type dengue fever virus according to claim 1, is characterized in that, described RT-LAMP reaction solution contains: 10mM dNTP, 10 × ThermoPol reaction buffer, 150mM MgSO
4solution, 5mM trimethyl-glycine, the volume ratio of four is 6 ~ 8:4 ~ 5:2 ~ 3:8 ~ 10.
5. the RT-LAMP test kit for Rapid identification 4 type dengue fever virus according to claim 1, is characterized in that, described developer is SYBR GREEN I.
6. the RT-LAMP test kit for Rapid identification 4 type dengue fever virus according to claim 1, is characterized in that, described positive control is the plasmid containing 4 type dengue fever virus NS1 gene fragments (SEQ ID NO.7), and negative control is DEPC water.
7. a method for Rapid identification dengue fever virus 4 type, is characterized in that, adopt the test kit described in any one of claim 1 ~ 6, add template ribonucleic acid, at 60 ~ 65 DEG C, isothermal duplication 60 ~ 90 minutes, specifically comprises the steps:
(1) template ribonucleic acid is extracted;
(2) ring mediated isothermal gene amplification reaction: 25 μ l reaction systems contain: each 7 ~ 9pmol/ μ l of inner primer 1,2, each 1 ~ 2 pmol/ μ l of outer primer 1,2, each 3 ~ 5 pmol/ μ l of ring primer 1,2, reaction solution 12 ~ 13 μ l, archaeal dna polymerase 7 ~ 9U, RNA 1 ~ 100ng to be checked, reversed transcriptive enzyme 1 ~ 2U, with sterilizing deionized water polishing to 25 μ l, then add the sealing liquid of 18 ~ 22 μ l, by above-mentioned reaction tubes in 60 ~ 65 DEG C of reaction 60 ~ 90min;
(3) result judges: in above-mentioned reaction tubes, add 1-2 μ l developer 10 × SYBR GREEN I, and the color of observing response liquid after mixing, if present green, be singapore hemorrhagic fever 4 C-type virus C, orange is then non-singapore hemorrhagic fever 4 C-type virus C.
8. method according to claim 7, it is characterized in that, 25 μ l reaction systems of ring mediated isothermal gene amplification reaction contain: each 8pmol/ μ l of inner primer 1,2, the each 1 pmol/ μ l of outer primer 1,2, each 4 pmol/ μ l, the reaction solution 12.5 μ l of ring primer 1,2, archaeal dna polymerase 8U, RNA 1 ~ 100ng to be checked, reversed transcriptive enzyme 1U, with sterilizing deionized water polishing to 25 μ l.
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| CN201510282511.1A CN104946792A (en) | 2015-05-28 | 2015-05-28 | RT-LAMP kit for rapidly identifying dengue virus type 4 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110714096A (en) * | 2019-10-17 | 2020-01-21 | 深圳市疾病预防控制中心 | Kit for simultaneously detecting 4 dengue fever virus types |
| CN112063762A (en) * | 2020-10-18 | 2020-12-11 | 宁波市疾病预防控制中心 | Rapid detection method for dengue virus |
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| US20110306036A1 (en) * | 2010-06-10 | 2011-12-15 | Allison Dauner | RT-LAMP assay for the detection of pan-serotype dengue virus |
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2015
- 2015-05-28 CN CN201510282511.1A patent/CN104946792A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110306036A1 (en) * | 2010-06-10 | 2011-12-15 | Allison Dauner | RT-LAMP assay for the detection of pan-serotype dengue virus |
Non-Patent Citations (2)
| Title |
|---|
| MANMOHAN PARIDA, ET, AL.: "Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription–Loop-Mediated Isothermal Amplification Assay", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| Y. SUWANWONG, ET. AL.: "Detection of dengue virus by simple RT-PCR using universal degenerate primers Observations from a preliminary study", 《ARCHIVES OF HELLENIC MEDICINE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110714096A (en) * | 2019-10-17 | 2020-01-21 | 深圳市疾病预防控制中心 | Kit for simultaneously detecting 4 dengue fever virus types |
| CN112063762A (en) * | 2020-10-18 | 2020-12-11 | 宁波市疾病预防控制中心 | Rapid detection method for dengue virus |
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Application publication date: 20150930 |