CN100494402C - Liquid phase chip for detecting Avian influenza virus H5N1 subtype - Google Patents
Liquid phase chip for detecting Avian influenza virus H5N1 subtype Download PDFInfo
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Abstract
The present invention relates to liquid phase chip method of detecting bird flu virus H5N1 subtype. The liquid phase chip technology includes the homogeny analysis on all the H5 and N1 serum subtype nucleic acid sequence capable of being searched in the nucleic acid sequence bank by means of biological informatic means to find out conservative region, design degenerate primer and specific probe on the H5 and N1 gene fragment and couple the specific probe with fluorescent coding microsphere to make specific detecting microsphere as the liquid phase chip; and recognizing the H5 and N1 gene fragment specifically with the liquid phase chip through two rounds of PCR reaction and reading out the detection result in the chip detection instrument. The method can detect bird flu virus H5N1 subtype quickly for early diagnosis.
Description
Technical field
The present invention relates to a kind of liquid-phase chip that is used to detect bird flue virus H 5 N 1 subtype.
Background technology
Influenza is one of common acute respiratory transmissible disease, because influenza virus very easily makes a variation, usually causes the whole nation even worldwide being very popular.Especially the public health emergencies such as appearance of the popular and human and bird fluenza case of nearly section time domestic and international some areas bird flu, all an urgent demand is made in early days laboratory diagnosis fast to above-mentioned disease.
Influenza virus belongs to orthomyxoviridae family, can be divided into first, second, the third three types.Structure and the different of genetic characteristics according to influenza A virus surface antigen hemagglutinin antigen (HA) and neuraminidase antigen (NA) can be divided into some hypotypes again, so far the HA that has been found that influenza A has 16 hypotypes (H1-H16), and NA has 9 hypotypes (N1-N9).Because the antigenicity of HA and NA morphs easily, so caused the constantly popular of influenza virus, therefore the evaluation for its type is crucial in the testing process of influenza virus.
The detection of influenza virus mainly contains four major parts: virus culture separation, serodiagnosis, virus antigen detect, viral nucleic acid detects.Wherein virus culture is separated, serology detects and somatotype is conventional method, but this class methods spended time is long, can't detect and somatotype influenza virus fast; And in the detection and somatotype of viral nucleic acid, a lot of special methods have fast appearred.The particularly application of round pcr is being brought into play increasing effect for influenza virus evaluation and somatotype.These methods are as RT-polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR), multiple reverse transcription polymerase chain reaction (multiplex reverse transcription-PCR, MRT-PCR), enzyme is exempted from PCR (PCR-enzymeimmunoassay, PCR-EIA), PCR in real time (real-time PCR, RT-PCR) etc. the speed and the sensitivity that detect have been improved, yet, although increasing method is used to detect influenza virus, there is not a kind of method to carry out somatotype fast and accurately as yet to the various hypotypes of influenza virus.Therefore set up that a kind of that the various types of influenza virus are carried out the method for Rapid identification is just very important.
(Multi-Analyte Suspension Array, MASA) technology is a kind of novel gene chip technology to liquid-phase chip.The core of this technology be fluorescence-encoded latex particle respectively with specific detection probes coupling, in suspension, add micro-sample then and combine with the specific detection particulate, the bonded result is noted with data mode by computer after the laser interpretation.This technology has high-throughput, highly sensitive, fireballing characteristics, and needed specimen amount is few, in aspect widespread uses such as gene type, tissue typing, infectious diseases detections.
Summary of the invention
Technical problem to be solved by this invention is: a kind of liquid-phase chip that is used to detect bird flue virus H 5 N 1 subtype is provided, so that bird flue virus H 5 N 1 subtype is carried out rapid detection and carries out early diagnosis, contribute for controlling the popular of bird flu and control and eliminating mutual infection of human and bird fluenza.
The technical solution adopted in the present invention is: described liquid-phase chip is a kind ofly to carry out the specific detection microballoon that coupling is made by specificity detection probe and fluorescence-encoded micro-beads; This liquid-phase chip can specific identification avian influenza virus H5 and N1 gene segment, its process is: following two kinds of primers (seeing Instructions Page 3 " specific detection primer " and " universal primer of biotin modification ") are used for two-wheeled PCR reaction, the PCR reaction product with mix microballoon hybridization after, read detected result by liquid-phase chip detector (Luminex) again.
Liquid-phase chip provided by the invention is used to detect bird flue virus H 5 N 1 subtype.
Main effect of the present invention is as follows:
We have designed at four pairs of Auele Specific Primers of avian influenza virus H 5 N 1 blood serum subtype and probe, and wherein H5 and N1 are divided into two groups (H5-1 and H5-2, N1-1 and N1-2) respectively; Use four kinds of fluorescence-encoded micro-beads, utilize liquid-phase chip technology that H5N1 hypotype and other common hypotype (A1, A2, A3, H9N2) sample have been carried out check and analysis.Discovery has than high specific, sensitivity and characteristics fast based on the bird flue virus H 5 N 1 subtype detection kit of liquid-phase chip technology development, bird flue virus H 5 N 1 subtype is being carried out rapid detection and is carrying out having important use value aspect the early diagnosis.
Utilize a kind of novel biochip technology---liquid-phase chip technology, set up and a kind of bird flue virus H 5 N 1 subtype is carried out the novel method of quick diagnosis, and make biological articles such as detection kit.Because this biological article has fast and higher specific characteristics, can rapid detection go out the H 5 N 1 avian influenza hypotype, thus can be for the control bird flu popular, and for control with eliminate human and bird fluenza and infect mutually and contribute.
In view of be used at present that method that bird flu detects can't the be fast and convenient various hypotypes of influenza virus are distinguished, based on above-mentioned effectiveness, the present invention for develop detect H5N1 hypotype and other various subtype influenza viruses more fast and effectively method a more wide prospect is provided.
Description of drawings
Fig. 1 is the RT-PCR of a routine H5N1 hypotype and the synoptic diagram of PCR product.Among the figure: DL2000 is Marker, and each electrophoretic band has been represented PCR product separately.
Fig. 2 to Fig. 5 adopts the synoptic diagram of Luminex100 system to mix primer amplified production hybridization detected result, wherein:
Fig. 2 is determining of gate value, makes the peak value that detects fluorescence between the gate value of determining;
Fig. 3 has indicated four kinds of pairing positions of mixing microballoon;
The routine H5N1 hypotype sample of Fig. 4 for detecting;
Fig. 5 is the A1 blood subgroup sample.
X-coordinate represents to hybridize the average fluorescent strength of product among Fig. 4 and Fig. 5 two figure, and ordinate zou is represented employed probe link coupled microballoon, and an endogenous Control is wherein arranged.
Embodiment
The present invention is based on liquid-phase chip technology, set up a kind of novel method of the H5N1 subtype avian influenza virus being carried out rapid detection.In the detection to various samples, we find that the bird flue virus H 5 N 1 subtype detection kit based on the liquid-phase chip technology development has than high specific, sensitivity and characteristics fast.
The present invention utilizes liquid-phase chip technology, the utilization bioinformation is gained knowledge and relevant information biology software, all H5, the N1 blood serum subtype nucleotide sequence that can retrieve in the nucleotide sequence storehouse carried out homology analysis, find out conservative region, design degenerated primer and specific probe at avian influenza virus H5 and N1 gene segment, degenerated primer comprises the universal primer of specific detection primer and biotin modification, specific probe is made specific detection microballoon, i.e. liquid-phase chip by carrying out coupling with fluorescence-encoded micro-beads.Liquid-phase chip can specific identification avian influenza virus H5 and N1 gene segment, by two-wheeled PCR reaction, and reads detected result by the liquid-phase chip detector.Liquid-phase chip can the generate a reagent box etc. biological article.
The invention will be further described below in conjunction with example and accompanying drawing.
One. the design of sequential analysis, primer probe is synthetic
Download all A type influenza virus H5 and N1 segment nucleotide sequence from the Genbank of NCBI, filter out the complete nucleotide sequence that needs.Sequence is compared, according to nucleic acid homology the nucleotide sequence of retrieving is divided into different groups for each segment.
We are divided into two group: H5-1 and H5-2, N1-1 and N1-2 respectively with H5 and N1 hypotype among the present invention.For each group, analyze and find out conserved sequence, then according to conserved sequence design primer and probe.Because single nucleic acid variance ratio is more, we use degenerated primer.Designed probe will be carried out coupling with the microballoon that uses in the chip, and 5 ' end needs amination to handle.
During the design primer, respectively add one section sequence (this sequence and influenza virus sequence not homology) by analysis at 5 of all forward primers and reverse primer ' end, be universal primer (Uni-forward) 5 '-tac agt cgg tcg cgt gcc tc and (Uni-reverse) 5 '-ctg gtc cgt act tcc gag cg so that second take turns amplified reaction.
Second takes turns amplified reaction uses these two sections sequences (Uni-Primers) that add above, and wherein oppositely 5 of universal primer Uni-reverse ' end carries out mark with vitamin H (biotin).
Synthetic following primer of final design and probe:
Specific detection primer is as follows:
H5-1For： 5′-tac?agt?cgg?tcg?cgt?gcc?tcg?gac?aat?ttt?raa?rcc?raa?tg-3′
H5-1Rev： 5′-ctg?gtc?cgt?act?tcc?gag?cgt?tat?cgc?mcc?cay?tgg?ag-3′
H5-2For： 5′-tac?agt?cgg?tcg?cgt?gcc?tca?atg?gac?aaa?gtg?gaa?gaa?t-3′
H5-2Rev： 5′-ctg?gtc?cgt?act?tcc?gag?cga?agg?cata?ctr?gaa?ttt?ata?g-3′
N1-1For： 5′-tac?agtcgg?tcg?cgt?gcc?tcg?caa?ttc?atc?tct?ttg?ycc-3′
N1-1Rev： 5′-ctg?gtc?cgtact?tcc?gag?cgc?acc?cac?agg?aca?act?c-3′
N1-2For： 5′-tac?agt?cgg?tcg?cgt?gcc?tcc?tcy?cac?ttg?gar?tgc?ag-3′
N1-2Rev： 5′-ctggtc?cgt?act?tcc?gag?cgc?crt?tgtatt?tca?ata?cag?c-3′
The universal primer of biotin modification is as follows:
Uni-For： 5′-tac?agt?cgg?tcg?cgt?gcc?tc-3′
Uni-Rev： 5′-biotin-ctg?gtc?cgt?act?tcc?gag?cg-3′
Specificity detection probe is as follows:
H5-1Probe：?5′-NH2-tat?gca?tac?aaa?att?gtc?aag?aar?gg-3′
H5-2Probe：?5′-NH2-ccw?gaa?tat?gca?tac?aag?ata?gtt?aa-3′
N1-1Probe：?5′-NH2-aaa?tcc?aag?ggg?gat?gtg?ttt?gtt?rt-3′
N1-2Probe：5′-NH2-gtt?ggt?tga?caa?ttg?gwa?ttt?cyg?g-3′。
Two .RT-PCR and PCR
We have designed two-wheeled PCR reaction.Wherein first round reaction is RT-PCR, utilizes Auele Specific Primer to carry out single stage method RT-PCR.Reaction system is 25uL, wherein adds RNA enzyme inhibitors 5U, forward and reverse primer each (every primer final concentration is 0.6uM), RNA template 1uL in 5 * buffer 5uL, dNTP 1uL, Enzyme 1uL, each reaction, adds water and supplies the 25uL system.Use PTC one 200PCR instrument, condition enactment is 50 ℃ of 30min of reverse transcription, 95 ℃ of 15min of sex change, and 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 30sec carry out 35 circulations, and last 72 ℃ are extended 5min.Second to take turns reaction be that to utilize Uni-Primers be the PCR reaction that template is carried out with first round reaction product.Reaction system is 50uL, 10 * buffer5uL wherein, and dNTP 1uL, TaqDNA polysaccharase 1U, Uni-forward 1uL, Uni-reverse 1.5uL adds back RT-PCR reaction product 1uL, adds water and supplies the 50uL system.Reaction conditions is set at 95 ℃ of 10min of sex change, 94 ℃ of 30sec, and 60 ℃ of 30sec, 72 ℃ of 30sec carry out 35 circulations, and last 72 ℃ are extended 5min.Reaction product is put 4 ℃ and is treated that hybridization detects.
Three. the preparation of liquid-phase chip
1, probe and microballoon coupling
Take out 50uL (1 * 10
6Individual) microballoon that needs, supernatant discarded behind the centrifugal 1min30sec of 12000rpm adds MES (0.1M, pH 4.5) 8uL, mixing.Used probe is diluted to 20uM with MES, adds 2uL in coupling system, add EDC (10mg/mL) 1.25uL for the first time, lucifuge is placed 30min behind the mixing.Repeat to add EDC once.Respectively wash once with 500uLTween20 (0.02% V/V) and SDS (0.1% W/V), at last microballoon is suspended in again in 20uL TE (PH8.0) solution, uses hematimeter counting microballoon quantity (being scaled every uL microballoon number after counting the number of four jiaos of 4 big grids) behind the mixing.4 ℃ keep in Dark Place.The microballoon that mixes the good probe of coupling, the concentration of using 1.5 * TMAC to be diluted to every kind of microballoon is 150/uL.
2, the solution hybridization of amplified production
With product with mix microballoon and hybridize, crossbred is 50uL, wherein mixes microballoon 33uL, PCR product 5uL adds TE and replenishes volume to 50uL.Behind 96 ℃ of sex change 5min, 52 ℃ of hybridization 15min.Add 52 ℃ of hybridization of SA-PE 5min, machine testing in the wait.
3, (Luminex 100 for multi-functional streaming dot matrix instrument
TM) setting
It is Events:100 that instrument parameter is set; Min event:20.
Gate value determines: use Luminex100
TMDirectly read and use the not microballoon of coupling probe in this experiment, determine gate value size according to the peak value that instrument reads.
Four. the detection of sample
1, RT-PCR and PCR are to the checking of primer
Utilize 1.5% agarose gel electrophoresis, we have detected the product of two-wheeled amplified reaction respectively.
Fig. 1 is the RT-PCR product result of a routine H5N1 hypotype sample, wherein amplified production do not occur in the H5-2 group.Amplified in other group and expectation segment product of the same size.In the RT-PCR result that the application mix primer carries out, except that the H5-2 group, other estimates that big or small slice stopping pregnancy thing all occurs, and has also amplified big and less non-specific segment simultaneously.
Second taking turns the PCR product and also obtaining equifinality in detecting what utilize that Uni-Primers carries out.H5-2 group does not obtain the PCR product, and analyzing its reason is that the H5-2 group is mainly H5N2 hypotype sequence, and we used be the H5N1 sample.
Employed other hypotype sample of current experiment is not found tangible corresponding big or small amplified production through electrophoresis detection.Prove that the primer that we design has specificity and applicability preferably.
2, Gate value's determines
Use Luminex100
TMDirectly read and use the not microballoon of coupling probe in this experiment, triplicate, thus determine to need gate value is set at 9294～18441 in this experiment.Instrument detecting the results are shown in Figure 2.
3, specificity test-results
As Fig. 3-5, H5N1 sample and other several frequently seen blood serum subtype sample have been detected.In H5N1 hypotype sample detected, four kinds of microballoons had all obtained higher fluorescence intensity, positive result; In the A1 blood subgroup sample detected, the N1-2 microballoon had obtained higher fluorescence intensity (the average fluorescent strength value is 185.07); In other hypotype sample, various microballoon fluorescence intensities are all lower, negative result.
4, sensitivity test result
After measured, this method is 10pg to the limit of identification of H5N1 hypotype RNA.The results are shown in Table 2, along with successively decreasing of RNA detection limit, the fluorescence intensity level of detection also successively decreases gradually.When 10pg, have only H5-1 and N1-2 group that higher fluorescence intensity level (greater than 100), positive result are arranged.
Five. subordinate list
Sensitivity test result
Claims (3)
1. liquid-phase chip that is used to detect bird flue virus H 5 N 1 subtype is characterized in that described liquid-phase chip is a kind ofly to carry out the specific detection microballoon that coupling is made by specificity detection probe and fluorescence-encoded micro-beads;
Specificity detection probe is:
H5-1Probe： 5′-NH2-tat?gca?tac?aaa?att?gtc?aag?aar?gg-3′
H5-2Probe： 5′-NH2-ccw?gaa?tat?gca?tac?aag?ata?gtt?aa-3′
N1-1Probe： 5′-NH2-aaa?tcc?aag?ggg?gat?gtg?ttt?gtt?rt-3′
N1-2Probe： 5′-NH2-gtt?ggt?tga?caa?ttg?gwa?ttt?cyg?g-3′。
2. liquid-phase chip according to claim 1, it is characterized in that specificity detection probe and fluorescence-encoded micro-beads carry out the link coupled method and be: take out the 50uL fluorescence-encoded micro-beads, supernatant discarded behind the centrifugal 1min30sec of 12000rpm, adding concentration is that 0.1M, pH value are 4.5 MES 8uL, mixing; Used probe is diluted to 20uM with MES, adds 2uL in coupling system, add the EDC 1.25uL first time of 10mg/mL, 30min keeps in Dark Place behind the mixing; Repeat to add EDC once; Again with the concentration of 500uL be the Tween20 of 0.02% V/V and 0.1%W/V each washing of SDS once, then microballoon is suspended in again pH and is in 8.0 the 20uL TE solution, use hematimeter counting microballoon quantity behind the mixing; At last, 4 ℃ keep in Dark Place,
Mixing four kinds of couplings has the fluorescent microsphere of specificity detection probe, and the quantity of using 1.5 * TMAC to be diluted to every kind of microballoon is 150/uL.
3. liquid-phase chip according to claim 1 is characterized in that: with liquid-phase chip generate a reagent box.
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Cited By (1)
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| CN103131794A (en) * | 2011-12-05 | 2013-06-05 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Suspension chip method capable of quickly diagnosing A-type H1N1 influenza viruses |
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| CN101392298B (en) * | 2008-07-15 | 2011-08-31 | 江苏省疾病预防控制中心 | Liquid-phase chip for detecting flu and H5N1 avian influenza virus and specific detection primer and preparation method thereof |
| CN101392302B (en) * | 2008-09-28 | 2011-04-20 | 中国疾病预防控制中心病毒病预防控制所 | Flu/human avian influenza virus detection gene chip and production method and use |
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