CN104805220A - Novel bunyavirus LAMP detection method - Google Patents

Novel bunyavirus LAMP detection method Download PDF

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CN104805220A
CN104805220A CN201510222504.2A CN201510222504A CN104805220A CN 104805220 A CN104805220 A CN 104805220A CN 201510222504 A CN201510222504 A CN 201510222504A CN 104805220 A CN104805220 A CN 104805220A
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lamp
reaction
primer
detection method
novel bunyavirus
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董雪
王秋雨
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Disease Prevention And Control Center Of Shenyang City
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract

The invention discloses a novel bunyavirus LAMP detection method, which belongs to the field of pathogenic microorganism detection. The method comprises the following steps: designing an LAMP primer sequence, preparing an LAMP reaction solution, carrying out LAMP method nucleic acid amplification, and detecting an LAMP reaction product. The LAMP primer sequence is as follows: a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. By adopting the method, the weaknesses in the prior art can be overcome; the novel bunyavirus LAMP detection method is rapid, efficient, high in sensitivity, good in specificity, convenient to operate and low in cost.

Description

A kind of novel bunyavirus LAMP detection method
Technical field
The invention belongs to the pathogenic microorganism examination field, be specifically related to a kind of novel bunyavirus LAMP detection method.
Background technology
Bunyaviridae (Bunyaviridae) is that a class has coating minus-stranded rna virus, because the Bu Niyaweila (Bunyamwera) first from Uganda western part is separated to and gains the name, in definite designation in 1975.Bunyaviridae is a section maximum in arboviruses, and its member about has 350, and constantly finds newcomer.Most of viruses of this section circulate between natural arthropods-vertebrates, can make the about kind more than 60 that people and (or) animal are caused a disease.In the mankind's disease of natural focus caused by this coe virus, important has hemorrhagic fever with renal syndrome (HFRS), Hantavirus pulmonary syndrome (HPS), Rift Valley fever (RVF), Crimean-Congo hemorrhagic fever (CCHF, domestic title Xinjiang hemorrhagic fever, XHF) and sandfly fever (Sandfly fever) etc.
Tick passes disease obvious seasonality, and 4 ~ September is onset peak.Infected regions multidigit is in hilly country, shallow mountain, and luxuriant vegetation, hard tick activity is active, and people are attacked by it often.Crowd is to the general susceptible of this kind of transmissible disease, and morbidity mostly is peasant, in highly distributing.In recent years, in Henan of central plain area of China, Shandong, Anhui, Hubei, Jiangsu and and 12 provinces such as Liaoning, Zhejiang there occurs the media biology transmissible disease that nearly thousand examples cause because being bitten by tick, tens of people is dead.Most of patients symptom is slight, and minority is auld, autoimmune function low, the patients clinical symptom of affecting treatment adversely is critical.The symptom of patient with severe symptoms has high fever, headache, illusion, general malaise, Nausea and vomiting, eye socket pain, pain in the back, sore muscle and One's spirits are drooping etc., and with degradation symptom under white corpuscle, thrombocyte Progressive symmetric erythrokeratodermia.In year March in September, 2010 to 2011, China successively has 6 provinces to occur heating companion thrombocytopenic syndromes (severe fever with thrombocytopeniasyndrome, SFTS) case, wherein 36 routine deaths.In September, 2010, CDC sample of blood, drawn from Hubei and Henan Er Sheng acute phase case makes further laboratory examination, and group of fabric study is explored in many ways, finally announce to isolate a kind of novel bunyavirus on March 16th, 2011, called after SFTS bunyavirus, and complete this virus gene sequence mensuration and tetraploid rice, assert that this virus is the new virus of bunyaviridae Phlebovirus, cause the great attention of domestic and international medical circle.
Novel bunyavirus infection morbidity is anxious, the state of an illness is heavy, case fatality rate is high, simultaneously can by propagation such as contact patient bloods, and early stage express laboratory is made a definite diagnosis for early diagnosis, early stage isolation, early treatment, prevention and control in early days significant.The experimental animal model that novel bunyavirus infects is set up in research, further further investigation its infect and pathogenesis and epidemic, being the new task that investigator faces, is also the key of symptomatic treatment.
High conservative region design Auele Specific Primer and the probe of subviral S, M, L gene fragment of Novel cloth Buddhist nun are selected at present, qualitative or the detection by quantitative of viral nucleic acid is made by the blood sample of RT-PCR method to acute phase (fall ill 2 weeks in) patient, although round pcr specificity and susceptibility higher, but rely on the PCR instrument of high degree of accuracy, experimental cost is comparatively large, is subject to many restrictions when large sample crowd Site Detection and clinical application.Therefore, in the urgent need to developing the sensitivity made new advances, special, novel bunyavirus diagnostic method fast, easily.
LAMP method designs 4 primers for 6 sequence areas of goal gene, by will the gene samples of amplification be needed to mix with primer, the archaeal dna polymerase with strand-displacement activity and substrate etc., under constant temperature (60 DEG C-65 DEG C, generally select 60 DEG C, 63 DEG C or 65 DEG C) anti-rock 30-60min, just can only have the target nucleic acid molecule amplification stripping 10 of several copy 9-10 10molecular level, this reaction does not need to make DNA double chain become strand with high temperature, and whole reaction process only just can need complete in constant water bath box, and the end product of reaction is a kind of mixture, be made up of the DNA of the stem-ring structure of multiple different stem length, by observing by product white Pyrophosphate phosphohydrolase (Mg 2p 2o 7) presence or absence that precipitates, just can judge the amplification whether success of target gene, a step can complete the amplification of gene and the detection of product.
LAMP technology increases, and the size of the object nucleic acid fragment of detection is generally advisable with 200-300bp.General experiment flow comprises: in GenBank, retrieve goal gene fragment (and being determined the homology of this fragment and other species genes by BLAST), artificial or Photographing On-line primer sets, primer synthesize, determine reaction system, have the archaeal dna polymerase of strand-displacement activity (if carry out reverse transcription LAMP amplification, only need in the reagent of DNA cloning, then add reversed transcriptive enzyme) effect lower constant-temperature amplification and reaction result judgement etc.
LAMP method breaches molecular Biological Detection technology in the past needs expensive plant and instrument and the restriction of complex operations, only 1h is needed to the whole process obtaining result from collecting sample, and sensitivity is much higher than traditional detection method, can with the naked eye directly judge whether to obtain amplified production.LAMP technology is as a kind of quick, easy gene diagnosis method, and in Novel cloth Buddhist nun Asia is detected, the gene amplification of alternative PCR, has broad application prospects.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide a kind of rapidly and efficiently, highly sensitive, specificity good, easy to operate, cheap novel bunyavirus LAMP detection technique method.
A kind of novel bunyavirus LAMP detection method of the present invention, comprises the following steps: the detection of the preparation of LAMP primer sequences Design, LAMP reaction solution, LAMP method nucleic acid amplification, LAMP reaction product.
Described LAMP primer sequences Design: the target sequence choosing 221-450 at novel bunyavirus gene fragment conservative region, design 8 primers, candidate drugs is screened in list of primers, according to entropy sequence, obtain 4 Auele Specific Primers that novel bunyavirus LAMP detects: forward inner primer FIP, oppositely inner primer BIP, forward outer primer F3, oppositely outer primer B3;
Described forward inner primer FIP:3 ' Duan You F2 district composition, the F2c regional complementarity that F2 district and target gene 3 ' are held, 5 ' the Flc regional sequence held and target gene F1 complementation;
Described reverse inner primer BIP:3 ' end is made up of B2c region, the B2c regional complementarity that B2 district and target gene 3 ' are held, 5 ' the B1c regional sequence held and target gene B1 complementation,
Described forward outer primer F3, with target gene F3c section complete complementary,
Described reverse outer primer B3, with target gene B3c section complete complementary.
FIP:F1c-F2CAGCCCTGCAGTGCTGCTTCCTTTGGGTCTCCTGCTTAGC,
F3:AGCAGTAATGTGGTCCAGTG,
BIP:B1c-B2AGCCGACCAAATCATCATCCCATTGTCACGTCAGCCTTGC,
B3:ACCCTGCCAGTTAGCCTC。
The step of described reaction solution preparation is: add respectively in LAMP reaction tubes Tris ?HCl, Repone K, ammonium sulfate, magnesium sulfate, Triton X ?100, Bst archaeal dna polymerase, AMV reversed transcriptive enzyme, dNTPs, trimethyl-glycine, primers F IP, BIP, F3 and B3, DEPC water;
Application RNA extracts reagent and extracts testing sample RNA template, adds RNA template, negative control replaces with DEPC water in the reaction solution prepared, mixing;
Described LAMP reaction solution is finally: dNTPs 1 μ L, 0.8mol/l trimethyl-glycine 5 μ L, 8mmol/L magnesium sulfate 1 μ L of 10 × ThermoPol reaction solution 2.5 μ L, 1.4mmol/l, 40pmol/l FIP 1 μ L, 40pmol/l BIP 1 μ L, 5pmol/l F31 μ L, 5pmol/l B31 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, 5U/ μ L AMV reversed transcriptive enzyme 1 μ L, adds template ribonucleic acid 5 μ L, negative control replaces with DEPC water in the reaction solution prepared; Last with DEPC water fluid infusion to 25 μ L, mixing.
Described LAMP method nucleic acid amplification step is: the reaction system adding template ribonucleic acid is of short duration centrifugal after mixing, then puts into 62-65 DEG C of water-bath immediately, reaction 40-60min, then is placed in 80 DEG C of water-baths and acts on 5min termination reaction, observations.
The detection method of described LAMP reaction product is: naked-eye observation by product Pyrophosphate phosphohydrolase precipitates; The reaction that product is positive, then liquid in pipe is white opacity, and negative reaction is then without this phenomenon.
In experimentation of the present invention; Choose the target sequence of 221-450 at novel bunyavirus gene fragment conservative region, design 8 primers, in list of primers, screen candidate drugs, according to entropy sequence,
Target sequence
221 CTAAGCAGTA ATGTGGTCCA GTGGTCTCTT TGGGTCTCCTGCTTAGCACA GGAGCTAGCT
281 AGTGCCCTGA AGCAGCACTG CAGGGCTGGT GAGTTCATCATCAAGAAGCT GAAGTTCTGG 341 CCTATCTATG TCATTATCAAGCCGACCAAA TCATCATCCC ATATCTTCTT CAGCTTGGGG 401ATCCGCAAGG CTGACGTGAC AAGGAGGCTA ACTGGCAGGGTCTTCTCTGA
Primer information
Obtain 4 Auele Specific Primers that novel bunyavirus LAMP detects: forward inner primer FIP, oppositely inner primer BIP, forward outer primer F3, oppositely outer primer B3.
Advantage of the present invention and positively effect as follows:
1, novel bunyavirus LAMP detection method strictly identifies 6 isolated areas on target nucleic acid sequence for the primer of 4 careful design, so can not be subject to the non-target sequences DNA impact existed in reaction mixture, has higher specificity.
2, the template amount of novel bunyavirus LAMP detection method amplification can be low to moderate lO copy or less, and product amplification index reached 10 9-10 10copy, high 1-2 the order of magnitude more general than common RT-PCR method, has higher sensitivity.
3, novel bunyavirus LAMP detection method does not need to be changed by temperature cycle to increase, thus exempts the time of alternating temperature process consumes, and the target gene of several copy just can be increased 10 by it in 60 minutes 9level, reaction fast.
4, novel bunyavirus LAMP detection method does not need temperature cycler and gel imaging system, only needs a water-bath, and detected result can direct visual perception, simple to operate, quick, be applicable to on-the-spot to use in a large number.
Embodiment
Agents useful for same of the present invention is as follows: LAMP method RNA amplification test kit (RNA Amplification Kit), Japanese Eiken Chemical (EIKEN CHEMICAL CO., LTD, Tochigi, Japan); LAMP reaction tubes (Reaction Tube), Japanese Eiken Chemical; 10 × ThermoPol reaction solution, Bst archaeal dna polymerase, AMV reversed transcriptive enzyme, Biolabs company; DNTPs, Pharmacia company; Trimethyl-glycine, Sigma company; Magnesium sulfate, Chemical Reagent Co., Ltd., Sinopharm Group; DNA Marker DL2000, Dalian Bao Bio-Engineering Company.PCR instrument, ABI company; Real-time turbidimeter La-320C, Japanese Eiken Chemical; Constant-temperature metal bath: Hangzhou BIOER Technology Co., Ltd; Electrophoresis apparatus, gel imaging system, Bio-Rad company; Spectrophotometer, NanoDrop ND-1000; .
Spectrophotometer is used to measure in the OD value at 260nm place the novel bunyavirus RNA solution that this laboratory is preserved, and according to once its concentration of formulae discovery: RNA concentration (μ g/ μ L)=OD 260× 40 × 10-3 × X, X is the extension rate of sample.
RNA is carried out RT-PCR amplification, and product is cut glue and is reclaimed after agarose gel electrophoresis observations, order-checking qualification.By to temperature of reaction, reaction times, Auele Specific Primer concentration, Adlerika concentration, the optimization of the conditions such as trimethyl-glycine concentration, set up Novel cloth Buddhist nun subviral LAMP reaction system 25 μ L, 10 × ThermoPol reaction solution 2.5 μ L is added in LAMP reaction tubes, the dNTPs 1 μ L of 1.4mmol/l, 0.8mol/l trimethyl-glycine 5 μ L, 8mmol/L magnesium sulfate 1 μ L, 40pmol/l FIP 1 μ, 40pmol/l BIP 1 μ L, 5pmol/l F31 μ L, 5pmol/l B31 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, 5U/ μ L AMV reversed transcriptive enzyme 1 μ L, template ribonucleic acid 5 μ L, DEPC water fluid infusion to 25 μ L, contrast with DEPC water belongs with yin, of short duration centrifugal after mixing, then 63.5 DEG C of water-baths are put into immediately, reaction 40min, be placed in 80 DEG C of water-baths again and act on 5min termination reaction, whether visual inspection liquid in pipe is in white opacity.
Get amplified production 5 μ L, detect in 1% agarose gel electrophoresis, confirm whether there occurs reaction.By visual results, the LAMP reaction liquid in pipe adding template ribonucleic acid is white opacity, and centrifugal rear adularescent is precipitated as positive reaction, and negative control pipe solution is then limpider clear solution.1% agarose gel electrophoresis result and LAMP react visual results and conform to.
The hemorrhagic fever virus preserved with this laboratory, yellow fever virus RNA are template, detect according to above-mentioned LAMP system, and contrast with novel bunyavirus template, get amplified production 5 μ L simultaneously and detect at 1% agarose gel electrophoresis, analyze the specificity of this reaction.By visual results, the LAMP reaction liquid in pipe adding novel bunyavirus RNA is white opacity, and centrifugal rear adularescent is precipitated as positive reaction, and the LAMP reaction tubes of hemorrhagic fever virus, yellow fever virus RNA is then limpider clear solution.1% agarose gel electrophoresis result and LAMP react visual results and conform to.
Novel bunyavirus RNA template is carried out series 10 × doubling dilution, and minimum extension rate is to 10 -5, and use spectrophotometric determination to dilute the RNA concentration of each gradient front, calculate RNA quality and be respectively 100ng, 10ng, 1ng, 0.1ng, 10pg, 1pg.The RNA 5 μ L getting each extension rate respectively carries out LAMP and RT-PCR reaction, utilizes 1% agarose gel electrophoresis and visual inspection LAMP result, determines the sensitivity of the method.The LAMP method detection sensitivity set up is 10 -4extension rate, can detect the RNA of 10pg.Judge the amplification of RT-PCR with 1% agarose gel electrophoresis, the sensitivity of RT-PCR is 10 -2extension rate, namely detects the RNA of 1ng.The sensitivity of visible LAMP is 100 times of RT-PCR.
Gather heating companion bleeding patients clinical sample 7 parts, determine wherein 1 part of positive through test in laboratory and clinical diagnosis, 6 parts of feminine genders.Extract test kit with RNA and extract viral RNA, rear LAMP method detects, and compares with 1% agarose gel electrophoresis detected result of RT-PCR detection method.The detected result of LAMP and RT-PCR is consistent, and recall rate is 100%, and this novel bunyavirus LAMP detection method is feasible.

Claims (5)

1. a novel bunyavirus LAMP detection method, is characterized in that comprising the following steps: the detection of the preparation of LAMP primer sequences Design, LAMP reaction solution, LAMP method nucleic acid amplification and LAMP reaction product.
2. according to the novel bunyavirus LAMP detection method of one according to claim 1, it is characterized in that: described LAMP primer sequences Design: the target sequence choosing 221-450 at novel bunyavirus gene fragment conservative region, design 8 primers, candidate drugs is screened in list of primers, according to entropy sequence, obtain 4 Auele Specific Primers that novel bunyavirus LAMP detects: forward inner primer FIP, oppositely inner primer BIP, forward outer primer F3, oppositely outer primer B3;
Described forward inner primer FIP:3 ' Duan You F2 district composition, the F2c regional complementarity that F2 district and target gene 3 ' are held, 5 ' the Flc regional sequence held and target gene F1 complementation;
Described reverse inner primer BIP:3 ' end is made up of B2c region, the B2c regional complementarity that B2 district and target gene 3 ' are held, 5 ' the B1c regional sequence held and target gene B1 complementation,
Described forward outer primer F3, with target gene F3c section complete complementary,
Described reverse outer primer B3, with target gene B3c section complete complementary;
FIP:F1c-F2CAGCCCTGCAGTGCTGCTTCCTTTGGGTCTCCTGCTTAGC,
F3:AGCAGTAATGTGGTCCAGTG,
BIP:B1c-B2AGCCGACCAAATCATCATCCCATTGTCACGTCAGCCTTGC,
B3:ACCCTGCCAGTTAGCCTC。
3. according to the novel bunyavirus LAMP detection method of one according to claim 1, it is characterized in that: the preparation steps of LAMP reaction solution is:
Tris-HCl, Repone K, ammonium sulfate, magnesium sulfate, Triton X-100, Bst archaeal dna polymerase, AMV reversed transcriptive enzyme, dNTPs, trimethyl-glycine, primers F IP, BIP, F3 and B3, DEPC water is added respectively in LAMP reaction tubes;
Application RNA extracts reagent and extracts testing sample RNA template, adds RNA template, negative control replaces with DEPC water in the reaction solution prepared, mixing;
Described LAMP reaction solution is finally: dNTPs 1 μ L, 0.8mol/l trimethyl-glycine 5 μ L, 8mmol/L magnesium sulfate 1 μ L of 10 × ThermoPol reaction solution 2.5 μ L, 1.4mmol/l, 40pmol/l FIP 1 μ L, 40pmol/l BIP 1 μ L, 5pmol/l F31 μ L, 5pmol/l B31 μ L, 8U/ μ L Bst archaeal dna polymerase 1 μ L, 5U/ μ L AMV reversed transcriptive enzyme 1 μ L, adds template ribonucleic acid 5 μ L, negative control replaces with DEPC water in the reaction solution prepared; Last with DEPC water fluid infusion to 25 μ L, mixing.
4. according to the novel bunyavirus LAMP detection method of one according to claim 1, it is characterized in that: described LAMP method nucleic acid amplification step is: the reaction system adding template ribonucleic acid is of short duration centrifugal after mixing, then 62-65 DEG C of water-bath is put into immediately, anti-40-60min, then be placed in 80 DEG C of water-baths and act on 5min termination reaction.
5. according to the novel bunyavirus LAMP detection method of one according to claim 1, it is characterized in that: the detection method of described LAMP reaction product is: naked-eye observation by product Pyrophosphate phosphohydrolase precipitates; The reaction that product is positive, then liquid in pipe is white opacity, and negative reaction is then without this phenomenon.
CN201510222504.2A 2015-05-05 2015-05-05 Novel bunyavirus LAMP detection method Pending CN104805220A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102618669A (en) * 2012-04-12 2012-08-01 中国人民解放军***联勤部疾病预防控制中心 Kit for quickly examining loop SFTSV (severe fever with thrombocytopenia syndrome bunyavirus) mediated isothermal amplification of severe fever with thrombocytopenia syndrome bunyavirus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102618669A (en) * 2012-04-12 2012-08-01 中国人民解放军***联勤部疾病预防控制中心 Kit for quickly examining loop SFTSV (severe fever with thrombocytopenia syndrome bunyavirus) mediated isothermal amplification of severe fever with thrombocytopenia syndrome bunyavirus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
包红梅: "《H5亚型禽流感病毒LAMP快速检测方法的建立》", 《中国预防兽医学报》 *

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Application publication date: 20150729