CN102108418B - Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses - Google Patents
Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses Download PDFInfo
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Abstract
本发明涉及一种采用环介导恒温核酸扩增(LAMP)技术对甲型H1N1流感病毒进行检测的方法。该检测方法包括：病毒RNA提取；逆转录及LAMP扩增；LAMP扩增产物单链化；采用核酸纳米金生物传感器对LAMP扩增产物的检测。本发明还提供了用于检测甲型H1N1流感病毒的试剂盒。本发明利用经特殊设计的LAMP内引物通过LAMP扩增过程将一段酶切位点序列引入到扩增产物中，然后使用限制性内切酶TspR I消化扩增产物并通过物理骤冷的办法获得大量长度为140bp左右的单链DNA产物，由于采用了特定引物的环介导恒温核酸扩增技术并结合含有特定探针的核酸纳米金生物传感器，使得所述的检测方法和试剂盒特异性高、检测迅速。
The invention relates to a method for detecting type A H1N1 influenza virus by adopting loop-mediated constant temperature nucleic acid amplification (LAMP) technology. The detection method comprises: extraction of viral RNA; reverse transcription and LAMP amplification; single-strandization of the LAMP amplification product; detection of the LAMP amplification product by using a nucleic acid nano-gold biosensor. The invention also provides a kit for detecting influenza A (H1N1) virus. In the present invention, a specially designed LAMP internal primer is used to introduce a restriction site sequence into the amplification product through the LAMP amplification process, and then the restriction endonuclease TspR I is used to digest the amplification product and obtain it by physical quenching. A large number of single-stranded DNA products with a length of about 140bp, due to the use of the loop-mediated constant temperature nucleic acid amplification technology with specific primers combined with the nucleic acid nano-gold biosensor containing specific probes, the detection method and kit are highly specific , Rapid detection.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of H1N1virus nucleic acid method for quick and testing product of being used for.
Background technology
Influenza is called for short influenza, is by first type, B-mode, acute respiratory transmissible disease that three kinds of influenza viruses of third type cause.Wherein, the host of influenza A virus is the widest, harm is maximum, and people, fowl, poultry all are the hosts of influenza A virus.According to its surface tissue (hemagglutinin H and neuraminidase N) and genetic characteristics is different; Influenza A virus can be divided into many hypotypes again; So far the blood clotting found of influenza A virus have 16 hypotypes (H1-H16), and neuraminidase has 9 hypotypes (N1-N9).
Infected the Influenza A H1N1 epidemic situation from Mexico outburst and the people that spreads to global a plurality of countries in 2009; Be because a kind of H1N1virus of new variation causes; Be called " public health emergency of international concern " by the World Health Organization, reached 6 grades of the highest alarm criteria.
This year, the popular Influenza A H1N1 was a hypotype in the influenza A virus, had spread to world many countries, less than one month, and the Influenza A H1N1 case that make a definite diagnosis in the whole world has broken through ten thousand examples, and many cases Introduced cases influenza case has also appearred in China.Caused certain harm for human life's safety, comprise that active and effective prevention and control measure has all been taked in the countries in the world of the Chinese government, with the rapid spread that wards off disease.In the face of the epidemic disease of unexpected outburst, carrying out disease prevention, diagnosis, pathogenesis and treatment research is the emphasis that association area is paid close attention to and studied.
Influenza A virus is a RNA viruses, and traditional technique of gene detection has Northern hybridization, Southern hybridization, Western blotting, PCR etc., and these detection methods not only waste time and energy, and poor specificity.In more than ten years in the past, real-time fluorescence quantitative PCR has developed into a kind of important function of gene detection technique, has highly sensitive and specificity, but needs expensive instrument and professional's operation.
Ring mediated constant temperature nucleic acid amplification (loop-mediated isothermal amplication of nucleic acid; Be called for short LAMP) techniques make use Bst deoxynucleotide polysaccharase and the special inside and outside primer of two couples that designs according to different target sequences; Six isolated areas on the specific recognition target sequence start the endless chain replacement(metathesis)reaction.This technology is a kind of ideal means of on the PCR basis, creating.The ring mediated constant temperature nucleic acid amplification technology is characterized in: 1. only need just ability amplified reaction of a steady temperature.Do not need special reagent, do not need to carry out in advance the sex change of double-stranded DNA; 2. high specific: use six sections, four primers, and the order of these six sections also has regulation.The specificity of LAMP method amplification is very high on the principle, and whether the existence that can judge target gene according to whether increasing, promptly can carry out the qualitative detection of bacterium or virus; 3. fast efficient amplification: amplification can be accomplished less than 1h, and productive rate is high.If on primer, further improve again, can improve its amplification efficiency greatly, proliferation time reduces on original basis; 4. highly sensitive: amplification template can reach 10 copies also or still less, detects linearity range 5 one magnitude; And researchist or testing staff's actually operating are very easy.The LAMP method is a kind of gene amplification method of easy, quick, high degree of specificity.And do not need special reagent and plant and instrument, therefore can set up the cheap detection architecture of total cost.This method is applicable to on-the-spot the detection and a large amount of sample high throughput testing, and at clinical disease diagnosis, fields such as gene chip exploitation and food hygiene quality check have broad application prospects.Through constant temperature gene amplification technology and round pcr (comprising the fluorescence real-time quantitative PCR technology) are compared; Can find and technology on methodology indexs such as sensitivity, specificity and sensing range, to be equivalent to or to be superior to round pcr; And do not rely on any special plant and instrument and realize on-the-spot high-throughput rapid detection, and detect cost far below fluorescent quantitative PCR technique.
The DNA nanotechnology of rising in recent years is to be principle with DNA base strictly complementary paired characteristic, is mainly used in the assembling of molecule, produces the assembling aggregation that function is arranged.The DNA nanotechnology comprises outside the gene chip, and also having a kind of is DNA to be attached to constitute nano particle gene probe on the nano particle, through and target DNA between complementation realize the assembling of nano particle, this has become a kind of novel DNA detection system.Utilization gold nano grain gene probe such as Mirkin and the hybridization of synthetic target form tangible aggregate under the transmission electron microscope, can judge that whether synthetic target exists (Chad A.Mirkin; R.L.L.; Robert C.Mucic &James J.Storhoff Nature 1996,382,607-609).Preparation such as Wang gold nano grain hepatitis B virus (HBV) DNA gene probe; Visualizing detects polymerase chain reaction,PCR product (the Wang YF of HBV DNA on slide; Pang DW, Zhang ZL, et al.Visual gene diagnosis of HBV andHCV based on nanoparticle p robe amp lification and silver staining enhancement.J Med Virol; 2003,70:2052211).Practise application Fe3O4 (nuclear)/Au (shell) nano particle HBVDNA gene probes such as east, through its application (Dong Xia, X.L. in detecting HBV DNA of hybridization research in dot hybridization on the nylon membrane or the liquid phase; Qin Ninga; Qianghua Lud, Kailun Yao, Zuli LiudJournal of Nanjing Medical University 2007; 21,207-212).But all there is shortcoming separately in above-mentioned detection technique; Like the transmission electron microscope of the Technology Need complex and expensive of Mirkin, Wang needs expensive import slide, and needs long prehybridization, hybridization washing process; Need complicated for a long time prehybridization when practising the detection in east, hybridize, wash film silver and dye process; Do not have the technological merit time and effort consuming, and all be in the experimental study stage, do not have commercialization.
So still need seek quick, sensitive, low-cost, the easy detection of nucleic acids instrument of operation.For cooperating this research; Studied the detection method of the nucleic acid nano-gold biosensor that can accurately detect H1N1virus rapidly and developed its test kit, reached the bamboo telegraph of control disease effectively for early stage extensive examination and have great importance.
Summary of the invention
The objective of the invention is to overcome the problem and shortage of existing H1N1virus detection technique and product existence, a kind of method and test kit that is used for rapid detection H1N1virus (popular new variant in 2009) is provided.
A kind of ring mediated constant temperature nucleic acid amplification technology that adopts of the present invention may further comprise the steps the method that H1N1virus detects:
1) extraction of viral RNA:
A) from the virus-culturing fluid of deactivation, get 200-400 μ l Virus Sample and add 0.5ml Trizol, concussion repeatedly, suction is beneficial to virolysis, hatches 5 minutes down for 20-37 ℃;
B) in employing virus cracking liquid, add the chloroform of 0.1ml and shaking to chylosis, 20-37 ℃ of hatching 10 minutes down under 4 ℃ of 12000rpm centrifugal 15 minutes, got supernatant;
C) in supernatant, add the Virahol of 0.1ml, 20-37 ℃ of hatching 10 minutes down, under 4 ℃ of 12000rpm centrifugal 15 minutes, remove supernatant, make deposition dry;
D) add the 75% washing with alcohol deposition of 0.5ml, under 4 ℃ of 7500rpm centrifugal 5 minutes, eliminate supernatant, make deposition dry, this is precipitated as the RNA of Virus Sample;
E) with diethylpyrocarbonate water 20 μ l dissolving RNA deposition, RNA solution is preserved down in-80 ℃;
2) rt and LAMP amplification
The rt that adopts and the reaction system of ring mediated constant temperature nucleic acid amplification; Comprise: 10 times of ring mediated constant temperature nucleic acid damping fluid, dNTP solution, RNA enzyme inhibitors, mg ion, Bst enzyme, ThermoScript II, upper reaches outer primer and downstream outer primer, upper reaches inner primer and downstream inner primers, and viral sample RNA; Wherein, The upper reaches outer primer of ring mediated constant temperature nucleic acid amplification reaction contains just like the nucleotide sequence shown in the SEQ ID NO.1; The upper reaches inner primer of ring mediated constant temperature nucleic acid amplification reaction contains just like the nucleotide sequence shown in the SEQ ID NO.2; The downstream outer primer of ring mediated constant temperature nucleic acid amplification reaction contains just like the nucleotide sequence shown in the SEQ ID NO.3, and the downstream inner primer of ring mediated constant temperature nucleic acid amplification reaction contains just like the nucleotide sequence shown in the SEQ ID NO.4;
The response procedures of ring mediated constant temperature nucleic acid amplification: 63 ℃ of water-baths 60 minutes;
3) the LAMP reaction product of single stranded gained:
A) to 2) to add 1 μ l concentration in the reaction product of gained be the restriction enzyme TspR I of 8U/ μ l, and water-bath 2 hours under 63 ℃ of conditions;
B) a) products therefrom water-bath under 98 ℃ of conditions was placed rapidly 10 minutes on ice after 5 minutes;
4) adopt detection and the interpretation of result of nucleic acid nano-gold biosensor to the reaction product of the ring mediated constant temperature nucleic acid amplification handled through single stranded:
Described nucleic acid nano-gold biosensor comprises the sample pad, spun glass, nitrocellulose filter and the thieving paper that from left to right are fixed in successively on the offset plate;
Scribble the nano gold mark oligonucleotide probe on the said spun glass, this oligonucleotide probe forms by sulfydryl modification and with the Radioactive colloidal gold coupling, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ ID NO.5;
Be fixed with two kinds of oligonucleotide probes on the said nitrocellulose filter, this oligonucleotide probe forms with biotin labeling and with the Streptavidin coupling; The oligonucleotide probe that is fixed near thieving paper one end forms nature controlling line, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ ID NO.6; The oligonucleotide probe that is fixed near spun glass one end forms detection line, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ IDNO.7;
The reaction product of the ring mediated constant temperature nucleic acid amplification of handling through single stranded of step 3 gained is added dropwise to respectively in the well of described H1N1 nucleic acid nano-gold biosensor; The situation of detection line and nature controlling line of observing is carried out interpretation of result: redness appears in detection line and nature controlling line simultaneously, shows that test sample is positive; Nature controlling line occurs red, and redness does not appear in detection line, shows that test sample is negative.
According to detection method of the present invention, in the described nucleic acid nano-gold biosensor, nature controlling line and detection line are at a distance of 3-10mm; The said adjacent part that is fixed in sample pad, spun glass, nitrocellulose filter and thieving paper on the offset plate 1-5mm that overlaps each other; The particle diameter of said Radioactive colloidal gold is 8-100nm.
A kind of test kit that is used to detect H1N1virus of the present invention, it comprises:
The ring mediated constant temperature nucleic acid amplification reaction reagent, it comprises: 10 times of LAMP damping fluids, mg ion, enzyme mixed solution, RNA enzyme inhibitors, upper reaches outer primer, upper reaches inner primer, downstream outer primer, downstream inner primer; Wherein, Upper reaches outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.1; Upper reaches inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.2; The downstream outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.3, and the downstream inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.4; Said enzyme mixed solution is mixed with volume ratio by Bst enzyme 8U/ μ l and reversed transcriptive enzyme 5U/ μ l and forms at 1: 1; And nucleic acid nano-gold biosensor.Described nucleic acid nano-gold biosensor comprises the sample pad, spun glass, nitrocellulose filter and the thieving paper that from left to right are fixed in successively on the offset plate; Scribble the nano gold mark oligonucleotide probe on the said spun glass, this oligonucleotide probe forms by sulfydryl modification and with the Radioactive colloidal gold coupling, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ ID NO.5; Be fixed with two kinds of oligonucleotide probes on the said nitrocellulose filter, this oligonucleotide probe forms with biotin labeling and with the Streptavidin coupling; The oligonucleotide probe that is fixed near thieving paper one end forms nature controlling line, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ ID NO.6; The oligonucleotide probe that is fixed near spun glass one end forms detection line, and this oligonucleotide probe comprises the nucleotide sequence shown in SEQ ID NO.7.
According to test kit of the present invention, said outer primer and the concentration ratio between the inner primer that is used for the ring mediated constant temperature nucleic acid amplification reaction is 1: 1～1: 10, is preferably 1: 4.Under this concentration ratio, the starting amplification procedure that is guided by outer primer can effectively promote the generation by the nucleic acid amplification product with repeat amplification protcol section structure of inner primer guiding; Under this concentration ratio condition, the generation of primer dimer can be effectively suppressed simultaneously, thereby makes LAMP amplified reaction efficient reach maximum.
According to test kit of the present invention; Saidly be used for the upper reaches inner primer of ring mediated constant temperature nucleic acid amplification reaction and the sequence of downstream inner primer all contains one section 9nt restriction enzyme TspR I restriction enzyme site sequence; Wherein, Restriction enzyme site sequence in the inner primer of the upper reaches is 5-GACACTGGA-3, and the restriction enzyme site sequence in the inner primer of downstream is 5-GACACTGTC-3.In the amplification procedure of LAMP, the restriction enzyme site sequence is introduced in the amplified production.The LAMP amplified production is after enzyme is cut digestion, and it is the product about 140bp that the long-chain of its repeating structure will be cut into length.Single stranded handle the oligonucleotide chain of this product gained can be effectively with biosensor in probe generation match reaction, thereby reach the target of detection.
The invention has the advantages that: the present invention combines surperficial synthetic chemistry, biological chemistry and physical chemistry; Gordian technique in solving the design of visualizing technique of gene detection and making; Utilize and a pair ofly through the LAMP amplification procedure one section restriction enzyme site sequence is incorporated in the amplified production through the LAMP of particular design inner primer; Use restriction enzyme TspR I digest amplification product and the way through the physics quenching to obtain the single stranded DNA product of a large amount of length about then as 140bp; Owing to adopted ring mediated constant temperature nucleic acid amplification (LAMP) technology and bind nucleic acid nano-gold biosensor, made that described detection method and test kit specificity are high, detect rapidly.Viral RNA only need can obtain a large amount of target dnas after constant temperature carries out rt and LAMP amplification, whole amplification procedure only needs 1 hour.Above-mentioned product, single stranded is dripped in the well of nano-gold biosensor after handling; Can come judged result through observing detection line and nature controlling line after about 10 minutes; Do not need technical professional and expensive plant and instrument; Be used commercial RNA and extract test kit, the present invention can satisfy basic unit in time, outdoor, influenza A virus (2009 is popular) detects requirement rapidly.
Description of drawings
Fig. 1 shows the result of nano-gold biosensor test sample, is respectively A/ws/33 (H1N1) sample, A/hongkong/8/68 (H3N2) sample, A/P2/8/34 (H1N1) sample, Type B, H1N1virus sample from left to right.
Embodiment
Embodiment one: to the LAMP primer of H1N1virus and the design of probe
In NCBI http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/request.cgi, find out the PA gene fragment order of all influenza A viruss, through multiple ratio to finding out the conservative section of influenza A virus.Adopt PrimerExplorer on its conservative fragments, to design primer and probe.
Through PA gene fragment order, adopt PrimerExplorer design primer and probe to H1N1 (2009 is popular).Institute's designed primer and probe and all virus sequences are compared, find out the strongest primer of variability and probe.
The total length PA gene order of above-mentioned H1N1 (2009 popular) derive from NCBI (＞gi|238914637|gb|GQ244322.1|Influenza A virus (A/Guangdong/05/2009 (H1N1)) segment 3 polymerase PA (PA) gene, completecds)
Design result is:
The primer of H1N1virus and specific probe:
Upper reaches outer primer: TACAGCAGAAGTGTCCCA SEQ ID NO.1
Downstream outer primer: CCTTCCTTTTATAATGAACCCA SEQ ID NO.3
Upper reaches inner primer: SEQ ID NO.2
CACAGGATGCATTGAGCAAGGGACACTGGAGCTACTGAA?TACATAATGAAGGGA
Downstream inner primer: SEQ ID NO.4
GGATGACTTTCAGCTGATCCCAGACACTGTCTACAGGTTT?GTTTTCCGTCTT
Probe 1:5-ThioMC6-D-TGGTCCTACATTTGCTTATCAT SEQ ID NO.5
Probe 2:Biotin-5-AAAAAAAAAAATGATAAGCAAATGTAGGACCA SEQ ID NO.6
Probe 3:TTTTTTTTTTTCATCAATCAATCTTTTTCACTTT-3-Biotin SEQ ID NO.7
Relation between primer, probe and the template DNA three is: probe 1 between the upstream and downstream primer with institute test sample article in the template DNA specificity complementary, the partial sequence that probe 3 covers between the upstream and downstream primers, probe 1 is complementary with probe 2 specificitys.
Embodiment two: the preparation that is used to detect the nucleic acid nano-gold biosensor of influenza A virus of the present invention
1. the preparation of nanometer gold (Radioactive colloidal gold):
The HAuCL that in the round-bottomed flask of 500ML, adds 100ml 0.01%
4Solution is heated to boiling while stirring; In above-mentioned solution, add 2ml 1% Trisodium Citrate, become blueness in the solution 20s, become wine red behind the 60s, continue to boil 10min, stop heating and continue to stir 15min; Colloidal gold solution keeps in Dark Place for 4 ℃, and nanometer gold is identified through 520nm maximum absorbance value.
2. the preparation of gold mark oligonucleotide probe: with 100 μ l deionized water dissolving 1OD DNA-probes 1, join in 5 times of spissated colloidal gold solutions of volume, 4 ℃ 24 hours; The sealing of 10% bovine serum albumin added the SDS of NaCl and 1% after 30 minutes, spend the night to final concentration 0.1M and 0.01%, 4 ℃ respectively, 12000 rev/mins centrifugal 30 minutes, abandon supernatant, deposition contains 20mM Na with 100 μ l
3PO
4, 5%BSA, 0.25%Tween and 10% sucrose suspend again, process suspension-s.
3. the preparation of gold mark pad
The gold mark oligonucleotide probe of the present invention preparation is applied on the spun glass, and 37 ℃ of dryings 2 hours are processed gold mark pad, and are subsequent use.
4. the coupling of biotinylated probe and Streptavidin and conjugate is fixing
Streptavidin can combine and form key stable and difficult fracture through hydrophilic bond and Van der Waals force with vitamin H, so the present invention adopts DNA probe labeled with biotin and Streptavidin hybrid reaction, adopts a stroke film metal spraying appearance to be applied on the nitrocellulose filter.
For example, with the biotin labeled DNA-probe 2 of 10 μ l deionized water dissolving 1OD, add 5 μ l (2mg/ml) chain and sistomycocins, reaction adopt to be drawn a film metal spraying appearance and is applied on the nitrocellulose filter detection line after 1 hour under the room temperature, 37 ℃ of dryings two hours.
DNA-probe 3 adopts and is fixed on the nature controlling line with quadrat method.
5. the processing of sample pad
Spun glass soaked in hybridization solution (0.16%TritonX-100,0.2M Tris, 0.2M sodium-chlor, pH 8.0) after 4 hours, 37 ℃ of dry for standby.
6. the assembling of nucleic acid nano-gold biosensor
Be fixed in the nitrocellulose filter that is fixed with oligonucleotide probe, thieving paper, the spun glass that scribbles the nanoparticle labeled oligonucleotide probe, sample pad on the offset plate successively; The adjacent part 2mm that overlaps each other promptly obtains nucleic acid nano-gold biosensor of the present invention after cutting into wide 4mm.
Embodiment three: preparation and the detection method that is used to detect the test kit of H1N1virus nucleic acid nano-gold biosensor of the present invention
The test kit of H1N1virus nucleic acid nano-gold biosensor comprises following composition:
The LAMP reaction reagent, the H1N1virus nucleic acid nano-gold biosensor;
Wherein said LAMP reaction reagent comprises: 10 times of RT-PCR damping fluid 150 μ l, dNTP solution 150 μ l, mg ion 150 μ l, enzyme mixed solution 60 μ l, RNA enzyme inhibitors 30 μ l, upper reaches outer primer 30 μ l, downstream outer primer 30 μ l, upper reaches inner primer 30 μ l, downstream inner primer 30 μ l; Wherein, Upper reaches outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.1; The downstream outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.3; Upper reaches inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.2, and the downstream inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.4;
Said enzyme mixed solution is mixed with volume ratio by Bst enzyme 8U/ μ l and reversed transcriptive enzyme 5U/ μ l and forms at 1: 1.
Detect the preparation of H1N1virus LAMP reaction solution:
Reagent usage quantity μ l
10 * damping fluid 5
DNTP mixed solution (10mM) 2
MgCl2(50mM) 2
RNA enzyme inhibitors 1
Enzyme mixed solution 2
Upper reaches outer primer (10 μ M) 1
Downstream outer primer (10 μ M) 1
Upper reaches inner primer (10 μ M) 4
Downstream inner primer (10 μ M) 4
RNA extracting solution 4
DEPC water 6
TV 50 μ l
Establish following reaction conditions: 63 ℃ of water-baths 60 minutes.
Be the restriction enzyme TspR I of 8U/ μ l with adding 1 μ l concentration in the LAMP product of above-mentioned detection H1N1virus after reaction finishes, place 63 ℃ of water-baths 2 hours; Water-bath after 5 minutes placed 98 ℃ of water-baths of endonuclease reaction product rapidly 10 minutes on ice after finishing.To drip in the well of H1N1virus nucleic acid nano-gold biosensor through the product that as above single stranded is handled gained at last, can carry out the result and judge in about 10 minutes.
Embodiment four: the experiment of the method for detection influenza A virus of the present invention
1, strain and source thereof
A/ws/33 (H1N1), A/hongkong/8/68 (H3N2) is provided by Chinese Academy of Sciences's Guangzhou biological medicine and health research institute; A/PR/8/34 (H1N1), Type B are that clinical isolated strain is provided by Guangzhou Inst. of Respiratory Diseases; H1N1 is provided by centers for disease control and prevention of the united states (CDC).Can above strain information be passed through http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/select.cgi? Go=1 searches.
2, viral RNA extracts
A) 200-400 μ l sample adds 0.5ml Trizol (throat swab source or virus-culturing fluid), concussion repeatedly, and suction is beneficial to lysis, and hatching is 5 minutes under the room temperature.
B) add the chloroform of 0.1ml and shaking to chylosis, hatching is 10 minutes under the room temperature, and under 4 ℃ of 12000rpm centrifugal 15 minutes, get supernatant, be transferred to another 1.5ml EP pipe.
C) add the Virahol of 0.1ml, hatching is 10 minutes under the room temperature, and under 4 ℃ of 12000rpm centrifugal 15 minutes, remove supernatant, pipe is upside down on the thieving paper, air-dry a little.
D) add 75% ethanol (the water preparation that DEPC handles) the washing RNA of 0.5ml, under 4 ℃ of 7500rpm centrifugal 5 minutes, be inverted on the thieving paper, remove after the supernatant and left standstill 5 minutes under the room temperature.
E) with DEPC (diethylpyrocarbonate) water 20ul dissolving RNA.
F) measure RNA concentration and A260/A280.
3, rt and LAMP amplification
1) take out test kit (seeing embodiment three), thaw in 4 ℃, each reagent is in for use with preceding 1000 rev/mins of low-speed centrifugals 1 minute.
2) by following component preparation LAMP reaction solution (the reaction solution preparation should be carried out on ice).Detect the preparation of influenza A virus LAMP reaction solution:
Reagent usage quantity μ l
10 * damping fluid 5
DNTP mixed solution (10mM) 2
MgCl2(50mM) 2
RNA enzyme inhibitors 1
Enzyme mixed solution 2
Upper reaches outer primer (10 μ M) 1
Downstream outer primer (10 μ M) 1
Upper reaches inner primer (10 μ M) 4
Downstream inner primer (10 μ M) 4
RNA extracting solution 4
DEPC water 6
TV 50 μ l
3) LAMP reaction water-bath program is provided with
63 ℃ of water-baths 60 minutes.
4. detect and result's judgement
With adding 1 μ l concentration in the above-mentioned LAMP product is the restriction enzyme TspR I of 8U/ μ l, places 63 ℃ of water-baths 2 hours; Water-bath after 5 minutes placed 98 ℃ of water-baths of endonuclease reaction product rapidly 10 minutes on ice after finishing.To drip on the sample pad of nano-gold biosensor through the product that as above single stranded is handled gained at last, got final product judged result through the appearance situation of observing T line and C line in about 10 minutes.
Quality control standard:
1) it is effective that red line proof nano-gold biosensor appears in C line (nature controlling line).
2) whether T line (detection line) red line occurs, is the positive negative standard of differentiating.
Criterion as a result:
1) red line appears in the C line, and red line appears in the T line simultaneously, explains that test sample is positive;
2) red line appears in the C line, and red line does not appear in the T line simultaneously, explains that test sample is negative:
3) red line does not appear in the C line, explains that nano biological sensor lost efficacy.
A/ws/33 (H1N1), A/hongkong/8/68 (H3N2), A/PR/8/34 (H1N1), Type B influenza virus detect through nucleic acid nano-gold biosensor and to have only the C line red line to occur and red line does not appear in the T line; H1N1virus red line (Fig. 1) all occurs through H1N1 nucleic acid nano-gold biosensor detection T line, C line.The equal accord with expectation expectation of test-results.
According to above-mentioned detection method, can design the relevant detection test kit.
This test kit that is used to detect H1N1virus comprises: the ring mediated constant temperature nucleic acid amplification reaction reagent, and it comprises: 10 times of LAMP damping fluids, mg ion, enzyme mixed solution, RNA enzyme inhibitors, upper reaches outer primer, upper reaches inner primer, downstream outer primer, downstream inner primer; Wherein, Upper reaches outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.1; Upper reaches inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.2; The downstream outer primer contains just like the nucleotide sequence shown in the SEQ ID NO.3, and the downstream inner primer contains just like the nucleotide sequence shown in the SEQ ID NO.4; Said enzyme mixed solution is mixed with volume ratio by Bst enzyme 8U/ μ l and reversed transcriptive enzyme 5U/ μ l and forms at 1: 1; And nucleic acid nano-gold biosensor.
In this test kit, the outer primer and the concentration ratio between the inner primer that are used for the ring mediated constant temperature nucleic acid amplification reaction are 1: 1～1: 10, are preferably 1: 4.
Preferably; Saidly be used for the upper reaches inner primer of ring mediated constant temperature nucleic acid amplification reaction and the sequence of downstream inner primer all contains one section 9nt restriction enzyme TspR I restriction enzyme site sequence; Wherein, Restriction enzyme site sequence in the inner primer of the upper reaches is 5-GACACTGGA-3, and the restriction enzyme site sequence in the inner primer of downstream is 5-GACACTGTC-3.
Sequence table (SEQUENCE LISTING)
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| CN102952896B (en) * | 2011-08-26 | 2014-12-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | Universal loop-mediated isothermal amplification kit for detecting influenza A virus and application of universal loop-mediated isothermal amplification kit |
| CN102816865B (en) * | 2011-10-31 | 2014-04-02 | 武汉大学 | H1N1, PRRSV and CSFV quadruple detection kit |
| CN109266784B (en) * | 2018-07-27 | 2022-04-08 | 广东海洋大学 | A closed DNA fluorescent biosensor and its application in the detection of influenza A (H1N1) virus |
| CN109609603A (en) * | 2019-01-10 | 2019-04-12 | 首都医科大学附属北京儿童医院 | A method for detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification combined with nanobiosensing |
| CN109880896A (en) * | 2019-03-13 | 2019-06-14 | 中山大学 | A multiplex LAMP kit and detection method for rapid identification of specific typing of bacterial polymyxin resistance gene mcr |
| CN109897902B (en) * | 2019-04-11 | 2020-11-24 | 中国农业大学 | Test method and kit for rapid detection of donkey-derived components in food |
| CN110129484A (en) * | 2019-06-05 | 2019-08-16 | 济南大学 | A method for detection of influenza A virus based on gold-silver bimetallic nanoclusters enhanced fluorescence-binding loop-mediated isothermal amplification |
| CN112444546B (en) * | 2019-09-03 | 2022-07-01 | 华中农业大学 | A kind of electrochemiluminescence sensor for detecting oligonucleotide and preparation method thereof |
| CN111944925A (en) * | 2020-08-12 | 2020-11-17 | 深圳市儒翰基因科技有限公司 | Primer combination and kit for detecting H1N1 influenza A virus |
| CN113073152A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | LAMP primer, probe and kit for detecting influenza B virus |
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| WO2002033128A2 (en) * | 2000-10-18 | 2002-04-25 | Pharmasset Limited | Multiplex quantification of nucleic acids in diseased cells |
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