CN102586475B - Preparation method and application of gene chip for detecting important respiratory pathogenic viruses - Google Patents
Preparation method and application of gene chip for detecting important respiratory pathogenic viruses Download PDFInfo
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Abstract
The invention relates to a gene chip for detecting important respiratory pathogenic viruses. A preparation method of the gene chip comprises the following steps of: preparing a specific primer; preparing a virus specific probe; preparing an oligonucleotide chip; establishing an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) system; establishing a hybrid system; preparing a visual detection reagent; and establishing a developing method. The gene chip prepared by the invention can be used for simultaneously discriminating nine general respiratory pathogenic viruses comprising A and B type influenza viruses, parainfluenza viruses type 1 and 2, human metapneumovirus, respiratory syncytial virus, measles virus, rubella virus and mumps virus, provides a new solution for quickly detecting the general respiratory pathogenic viruses at high throughout and can provide guidance for monitoring, clinically diagnosing and treating the respiratory pathogenic viruses.
Description
Technical field
The present invention relates to preparation and purposes that nine kinds of common respiratory tract Causative virus detect gene chip, belong to the gene chip detecting technique field.
Background technology
Respirovirus refers to that a big class can invade that respiratory tract causes the respiratory tract local patholoic change or only is the invasion door with the respiratory tract, mainly causes the virus of the outer histoorgan pathology of respiratory tract.The virus of totally 239 types can cause acute respiratory infection to have at least more than 10 of 7 Viraceaes to belong at present.Acute respiratory infection is one of common clinical, has become a public health problem of serious threat human health.According to World Health Organization's statistical report in 2002, acute respiratory infection occupies in the ten big causes of death of the whole world the 3rd, and the whole world nearly four million peoples in every year die from acute respiratory infection.In children and adult, the acute respiratory infection more than 90% is all caused by virus.Modal Respirovirus has the first and second type influenza viruses (influenza virus), 1,2,3 type parainfluenza viruses (HPIV), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), Measles virus (Measlesvirus), mumps virus (Mumps virus), rubella virus (Rubella virus) etc.These viruses have characteristics such as infectivity is strong, propagation is fast, latent period is short, morbidity is anxious.Due to illness malicious type is of a great variety and its symptom is similar after the infection, as symptoms such as rhinitis, runny nose, nasal obstruction, cough, slight pharyngitis, whole body heatings, therefore is difficult to clinically differentiate.
Examination virus type promptly and accurately is the key link of clinical diagnosis and treatment and diseases monitoring.Traditional Respirovirus laboratory detection method comprises: the histocyte culture method; Serology and direct Detection Method (comprising electron microscopy); Direct immunofluorescence antibody act (IFA/DFA) indirectly; Enzyme immunoassay (EIA), enzyme assay (neuraminic acid enzymatic determination); Nucleic acid amplification (PCR).Separation and Culture and the traditional experiment technology such as evaluation and serology experiment of virus once played a part very important in the virus disease diagnosis.Histocyte is cultivated and is remained the important technical of finding new virus, is that other any technology are irreplaceable.Yet the defective that these technology all also exist some to remedy is during operational cost, effort, susceptibility is poor, diagnosis efficiency is low etc.In a word, traditional laboratory diagnostic method and the experimental technique of using at present can not satisfy the needs of clinical diagnosis far away, and it is recurrent that the doctor carries out the thing that virus disease diagnoses only according to own clinical experience.Accordingly, the laboratory diagnosis technology of virus disease must be reformed, and seek and set up quick, special, responsive and easy experimental technique, to adapt to the needs of clinical position.
Biochip technology is a kind of high-new biotechnology for genetic analysis that grows up early 1990s, is widely used in the research of clinical disease, especially for the infection of some microbial pathogenes of diagnosis.Compare with traditional Protocols in Molecular Biology, its outstanding characteristics of biochip technology are high-throughput, integrated, microminiaturized, automatization etc.Be particularly suitable for a large amount of unknown samples are carried out the analysis of parallel fast high-flux.The genome of virus is simpler, and every kind of virus has only a kind of nucleic acid, but virus multiplication speed is exceedingly fast, so virus has more genetic instability than other microorganisms, i.e. variability.The biochip technology of domestic and foreign literature patent report mostly is random primer in conjunction with method for gene chip for detection of the method for Respirovirus.Though the sensing range of random PCR TRAP has had very big change, but the target gene of this method amplification is used for chip hybridization, its hybridization signal poor specificity, domestic relevant research comprises the cDNA probe gene chip of preparations such as Yang Yinhui, can successfully detect 10 kinds of strong RNA viruses such as SARS virus, eastern equine encephalitis virus, its susceptibility is 10
2Copy number, but specificity is relatively poor, and between the probe of other genus non-specific hybridization is arranged; The gene chip that can detect tens kinds of Respiroviruses has been set up on Huangyan mountain etc., the long 60mer of specific specificity probe, sample amplification adopts joint PCR method at random, use this chip can successfully detect first type and the Influenza B virus of cell cultures, but specificity and sensitivity are all not high, because the primer does not have specificity when amplification, must cause amplification efficiency low yet, so also corresponding meeting decline of detection sensitivity.
Fluorescence detection is the conventional sense method of gene chip, is about to the fluorochrome compound and directly or indirectly is marked on the nucleic acid to be checked, and the fluorescent signal of generation adopts laser confocal scanning to detect.The shortcoming of this method: fluorescent signal is easily saturated, easily cancellation; There is the autofluorescence phenomenon; And the more important thing is that detecting instrument is expensive, and the volume heaviness, thus seriously limited gene chip applying at home, especially medium and small medical institutions and on-the-spot applying of detecting.It is imperative that cost of development gene chip cheap, easy to detect, that be fit to on-the-spot detection detects new technology.
The appearance of gold label silver stain technology (GLSS) has remedied the deficiency of fluorescence detection, makes the visual detection of gene chip become possibility.The principle of this technology is the PCR product of at first using nano gold mark to be checked, introduces silver ions then in system.Small-size effect makes nanometer gold have very strong catalytic reduction effect, silver ion reduction on every side can be become silver-colored particle; And the further silver ions around the catalytic reduction of these silver-colored particles.The katalysis of this cascade waterfall makes silver-colored particle more poly-more many, tightly wraps up nm gold particles and is accumulated into bulk silver shell, forms macroscopic black particle, and naked eyes can be observed.
Can significantly reduce the detection cost of biochip based on the visual chip detection technology of the principle of gold label silver stain, but the detection sensitivity of single step gold label silver stain can't satisfy the requirements at the higher level in Molecular Detection field, further improve visual sensitivity, make visual detection can replace fluoroscopic examination fully, significant for applying of biochip technology.TSA (tyramine siganal amplification, the tyrasamine signal amplification technique) be a kind of based on horseradish peroxidase (horseradishperoxidase, HRP) the bio signal amplifying technique of catalysis is proposed in 1989 first by people such as Bobrow.It is a phenolic group compound that signal amplifies the molecule tyrasamine, can be used as the effect substrate of HRP.The principle of TSA is under the HRP enzyme catalysis, connects haptenic tyrasamine molecule deposition in a large number, and these haptens mainly are small-molecule substances such as vitamin H, fluorescein.1992, the TSA system that Adams will use Biotin-Tyramine at first introduced immunohistochemical methods, was used for antigen or detection of antibodies.Now, the TSA technology has all obtained using widely at numerous areas such as immunoblotting, elisa assay and in situ hybridizations.
This research adopt multiplex PCR in conjunction with visual chip detection technical research important respiratory tract Causative virus detect gene chip kit, this chip agent box susceptibility is good, the specificity height is suitable for the Rapid identification of various respiratory road virus.
Summary of the invention
The object of the present invention is to provide a kind of visual gene chip that detects common respiratory tract Causative virus nucleic acid, this chip relies on visual chip detection technology to realize the visual of detection signal, can detect nine kinds of respiratory tract Causative virus simultaneously, comprise A, Type B influenza virus, parainfluenza virus 1,2 types, human metapneumovirus, respiratory syncytial virus, Measles virus, rubella virus, mumps virus, thus realize the purpose of high-throughput, special, responsive, the common respiratory tract Causative virus of rapid detection.
In order to achieve the above object, the present invention has developed the visual gene chip of common respiratory tract Causative virus detection of nucleic acids, and its preparation method is as follows:
1. step 1: preparation special primer
Through each Respirovirus genome of comparison, select special, the conservative fragments of virus as detecting target gene, under the prerequisite that guarantees each viral target gene specific amplification, take into account its sensitivity, so nine kinds of Respirovirus Auele Specific Primers are in charge of combination, have finally determined 3 pipe multiple RT-PCR systems through optimizing.Preferred 9 pairs of Respirovirus Auele Specific Primers and corresponding amplified target viral species thereof and be in charge of combined situation, as shown in table 1:
Table 1 Respirovirus Auele Specific Primer and corresponding amplified target viral species thereof and be in charge of combined situation
2. step 2: preparation virus-specific probe
In line with special principle between guarding between type, belonging to, according to the comparison between 9 kinds of Respirovirus target-gene sequences, the special relatively district of the sequence in upstream and downstream primer scope carries out the design of specific probe.Every kind of corresponding specific oligonucleotide probe of target virus.The target virus of preferred virus-specific probe sequence and correspondence is as shown in table 2:
Table 2 virus-specific probe sequence and corresponding target virus
3. step 3: preparation oligonucleotide chip
An embodiment preferred, each oligonucleotide probe is when point sample in the step 2, and (6 * SSC 0.1%SDS) is diluted to final concentration 50 μ M with 2 * sampling liquid.With commercially available gene chip sample applying instrument with probe points to the aldehyde radical modification slide of blank, the point sample amount of probe is 3nl.After the oligonucleotide chip preparation finishes, placed dry 18 hours in room temperature at least before the use.This chip features is to comprise in the oligonucleotide probe array nine kinds of Respirovirus specific probes, and is as shown in table 3.Wherein sheet matrix control probe is the 20T sequence, and 5 ' end cy3 mark, 3 ' end NH2 modify, and are used for monitoring aldehyde radical sheet sheet matrix amount.
Table 3 oligonucleotide probe array
| The control of sheet matrix | The control of sheet matrix | The control of sheet matrix | The control of sheet matrix | The control of sheet matrix | The control of sheet matrix |
| The control of sheet matrix | HMPV | Meales | Mump | Rubella | HRSV |
| The control of sheet matrix | HMPV | Meales | Mump | Rubella | HRSV |
| The control of sheet matrix | HMPV | Meales | Mump | Rubella | HRSV |
| The control of sheet matrix | PIV1 | PIV2 | INFA | INFB | Blank |
| The control of sheet matrix | PIV1 | PIV2 | INFA | INFB | Blank |
| The control of sheet matrix | PIV1 | PIV2 | INFA | INFB | Blank |
4. step 4: set up the RT-PCR system
The RT-PCR system is characterized as multiple asymmetric RT-PCR reaction system in the gene chip of the present invention.Suitable R T-PCR system can further improve the sensitivity of chip detection.Factors such as consumption to the absolute concentration of labeled primer and non-marked primer and relative proportion, enzyme are optimized.Preferred RT-PCR system, as shown in table 4:
Table 4RT-PCR system formulation
Preferred RT-PCR amplification condition is as shown in table 5:
The preferred RT-PCR amplification condition of table 5
5. step 5: set up the hybridization system
Suitable hybridization system also has great role to specificity and the sensitivity improving of chip.Hybridization solution composition, hybridization conditions and the post-hybridization washing condition of specificity and sensitivity have been obtained to guarantee simultaneously by optimization.The RT-PCR product mixes with the hybridization solution equal-volume in the hybridization system, and preferred each composition final concentration of hybridization solution is 9 * SSC, 0.3%SDS, 11% methane amide, 11%50 * Denhardt ' s.Preferred hybridization conditions is 45 ℃ of water-bath hybridization 1 hour.Preferred wash conditions be washing lotion A under the normal temperature (1 * SSC, 0.2%SDS), washing lotion B (0.2 * SSC) and washing lotion C (respectively wash 20s in 0.1 * SSC).
5. step 6: prepare visual detection reagent and set up coloration method
1. add the Streptavidin-HRP 10 μ l that press 1: 1000 dilution proportion at each hybridization region of chip, the diluent composition is 1 * PBS+0.1%BSA, places 30min for 37 ℃; (1 * PBS+0.05%Tween20) cleans 20s, and triplicate is put room temperature and dried with the PBST washing lotion to take out the back.2. add the Biotin-Tyramine 10 μ l that press 1: 500 dilution proportion at each hybridization region of chip, the diluent composition is 1 * PBS+0.1%BSA, places 30min for 37 ℃; Take out the back and clean 20s with the PBST washing lotion, triplicate is put room temperature and is dried.3. add the Streptavidin-Nanogold 10 μ l that press 1: 40 dilution proportion at each hybridization region of chip, diluent composition: 1 * PBS+0.1%BSA places 30min for 37 ℃; Take out the back and clean 20s with the PBST washing lotion, triplicate is used deionized water rinsing, puts room temperature and dries.4. with the silver-colored transfection reagent A liquid (aqueous solution of Silver monoacetate, concentration 4mg/mL) and B liquid (the citrate buffer solution solution of quinhydrones, concentration 10mg/mL) equal-volume mixes, A, the B mixed solution of each hybridization region lucifuge adding immediately 30ul, treat to occur on the chip stopping colour developing behind macroscopic grey or the black round dot, use washed with de-ionized water, dry.
More than the visual gene chip of Zhi Bei common respiratory tract Causative virus nucleic acid comprises oligonucleotide chip, RT-PCR system, hybridization solution, washing lotion A, washing lotion B, washing lotion C, Streptavidin-HRP, Biotin-Tyramine, Streptavidin-Nanogold, diluent, PBST washing lotion, silver-colored transfection reagent A liquid and B liquid.
An embodiment preferred is used commercially available RNA to extract test kit and is extracted viral RNA, and as the QIAamp viral RNA mini kit extraction viral RNA of Qiagen company, extraction is extracted the test kit specification sheets with reference to corresponding RNA and carried out.Extracting viral RNA uses the RT-PCR system to increase according to the amplification condition in the step 4.The RT-PCR product mixes with the hybridization solution equal-volume, adds in the oligonucleotide chip, hybridizes according to the hybridization conditions of step 5, washs according to the wash conditions of step 5, and the visible detection method according to step 6 develops the color again.
Chip after the colour developing can make and with the naked eye carry out interpretation, also uses commercially available visual chip scanner to scan, and operation analysis software sentence read result.
The present invention has set up a kind of gene chip based on visual detection method, can distinguish 9 kinds of common Respiroviruses, comprise influenza A, influenza B, Parainfluenza type 1 virus, 2 types, human metapneumovirus, respiratory syncytial, measles, rubella mumps virus.Performance is investigated and is shown that the present invention can accurately distinguish 9 kinds of common respiratory tract Causative virus, and can carry out accurate somatotype to 1 type, 2 types of A type, Type B and parainfluenza virus in the influenza virus, and specificity is good.The present invention all can detect 10 to 9 kinds of Respiroviruses
2The in-vitro transcription RNA of copies/ system.By the detection of respiratory virus infection patient suspected throat swab, method of the present invention and real-time fluorescence quantitative RT-PCR method have higher concordance rate.
Gene chip of the present invention and corresponding preparation method have stronger practicality.With conventional cultural method relatively, have quick, accurate, sensitive advantage, compare with other nucleic acid detection methods with immunological method, have high-throughput, advantage that specificity is high.Another outstanding advantage of the present invention is to need not to use expensive fluorescent scanning instrument, and as seen the signal naked eyes can be implemented in the application in basic medical unit and on-the-spot the detection.Gene chip of the present invention provides a kind of new solution for common respiratory tract Causative virus high-throughput, rapid detection, and the monitoring, clinical diagnosis and the treatment that can be the respiratory tract Causative virus provide guidance.
Description of drawings
Fig. 1: 9 kinds of Respirovirus specific amplification products agarose gel electrophoresis figure.M is molecular weight standard (stripe size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom) among the figure; 1 is Measles virus, amplified production length 600bp; 2 is rubella virus, amplified production length 321bp; 3 is mumps virus, amplified production length 406bp; 4 is human metapneumovirus, amplified production length 526bp; 5 is respiratory syncytial virus, amplified production length 260bp; 6 is influenza virus A type-H3N2 cloud, amplified production length 103bp; 7 is that influenza virus A type-H3N2 is dark, amplified production length 103bp; 8 is influenza virus A type-H1N1 Shanghai, amplified production length 103bp; 9 is Parainfluenza type 1 virus, amplified production length 128bp; 10 is acute laryngo-tracheo-bronchitis virus, amplified production length 116bp; 11 is influenza virus B type-Malaisia, amplified production length 144bp; 12 is influenza virus B type-Shandong, amplified production length 144bp; 13 is influenza virus B type-Fujian, amplified production length 144bp; 14 is influenza virus B type-Zhejiang, amplified production length 144bp; 15 is influenza virus B type-Tianjin, amplified production length 144bp; 16 negative contrasts.
Fig. 2: the probe array figure that the visual detection gene chip of important respiratory tract Causative virus is final.Round dot represents a point sample of probe among the figure, and 3 round dots of vertical direction are that 3 times an of probe repeat point samples.Round dot in the frame of broken lines is each Respirovirus specific probe, and the last item is blank.The outer round dot of frame of broken lines is identity column.
Fig. 3: the visual detection gene chip of important respiratory tract Causative virus nucleic acid specificity evaluation result figure.1 is human metapneumovirus among the figure; 2 is respiratory syncytial virus; 3 is Measles virus; 4 is mumps virus; 5 is rubella virus; 6 is the influenza virus A type; 7 is the influenza virus B type; 8 is Parainfluenza type 1 virus; 9 is acute laryngo-tracheo-bronchitis virus.
Fig. 4: the visual detection gene chip of important respiratory tract Causative virus sensitivity chip detection is figure as a result.Be example with human metapneumovirus and acute laryngo-tracheo-bronchitis virus.A1-G1 representative metapneumovirus sensitivity evaluation result among the figure wherein; A2-G2 represents acute laryngo-tracheo-bronchitis virus sensitivity evaluation result.A-G represents positive control, 10 successively
5Copies/ μ l, 10
4Copies/ μ l, 10
3Copies/ μ l, 10
2Copies/ μ l, 10
1Copies/ μ l and negative control.
Fig. 5: RT-PCR product agarose electrophoresis figure behind respiratory syncytial virus, the influenza virus B type gradient dilution.M:DL2000 molecular weight standard among the figure (stripe size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom); 1: virus-culturing fluid stoste; 2: 10 times of dilutions of virus-culturing fluid; 3: 100 times of dilutions of virus-culturing fluid; 4: 1000 times of dilutions of virus-culturing fluid; 5: 10000 times of dilutions of virus-culturing fluid; 6: 100000 times of dilutions of virus-culturing fluid.
Fig. 6: chip detection figure as a result behind respiratory syncytial virus, the influenza virus B type gradient dilution.Among the figure 1: respiratory syncytial virus nutrient solution stoste; 2: 10 times of dilutions of respiratory syncytial virus nutrient solution; 3: 100 times of dilutions of respiratory syncytial virus nutrient solution; 4: 1000 times of dilutions of respiratory syncytial virus nutrient solution; 5: 10000 times of dilutions of respiratory syncytial virus nutrient solution; 6: 100000 times of dilutions of respiratory syncytial virus nutrient solution; 7: influenza virus B type nutrient solution stoste; 8: 10 times of dilutions of influenza virus B type nutrient solution; 9: 100 times of dilutions of influenza virus B type nutrient solution; 10: 1000 times of dilutions of influenza virus B type nutrient solution; 11: 10000 times of dilutions of influenza virus B type nutrient solution; 12: 100000 times of dilutions of influenza virus B type nutrient solution.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
Embodiment 1: the development of the visual detection gene chip of important respiratory tract Causative virus
One, Auele Specific Primer design and screening
In order to realize the specific amplification to 9 kinds of Respiroviruses, use divides three pipe multiple RT-PCR systems to increase through 9 pairs of Auele Specific Primers of screening, primer sequence and corresponding target Virus Info see Table 1, the agarose electrophoresis of amplified production the results are shown in accompanying drawing 1, as seen from the figure, 9 kinds of Respiroviruses are all by specific amplification.
Two, the screening of specific probe
The non-specific intersection between probe and respiratory tract, other viruses of digestive tube at first got rid of in the screening of various probe, uses 9 kinds viral RNA to hybridize respectively as RT-PCR product and the alternative probe of template then, investigates each viral probe specificity.Finishing screen is chosen 9 specific probes.Probe sequence and corresponding target virus see Table 2.
Three, oligonucleotide chip preparation and probe array
After finishing probe screening, determine final probe array, seen Table 3 and accompanying drawing 2.Wherein first of the array row and first classifies the control of sheet matrix as, and blank is 2 * sampling liquid.
Four, RT-PCR system
The RT-PCR system is characterised in that and is multiple asymmetric RT-PCR reaction suitable R T-PCR system among the present invention.Suitable R T-PCR system can further improve the sensitivity of chip detection.Factors such as consumption to the absolute concentration of labeled primer and non-marked primer and relative proportion, enzyme are optimized.The RT-PCR system that system is finally determined see Table 4 and amplification condition see Table 5, this moment, the probe gray-scale value of reference material was stronger, and low copy template 10
3Copie/ μ l still can detect.
Five, foundation and optimization hybridization system
Hybridization solution composition, hybridization conditions and the post-hybridization washing condition of specificity and sensitivity have been obtained to guarantee simultaneously by optimization.The RT-PCR product mixes with the hybridization solution equal-volume in the hybridization system, and each composition final concentration of hybridization solution is 9 * SSC, 0.3%SDS, 11% methane amide, 11%50 * Denhardt ' s.Hybridization conditions is 45 ℃ of water-bath hybridization 1 hour.Wash conditions be washing lotion A under the normal temperature (1 * SSC, 0.2%SDS), washing lotion B (0.2 * SSC) and washing lotion C (respectively wash 20s in 0.1 * SSC).
Six, set up visible detection method
Prepared and diluted liquid, PBST washing lotion, diluent composition are 1 * PBS, 0.1%BSA, and PBST washing lotion composition is 1 * PBS, 0.05%Tween20.
Diluting following raw material: Streptavidin-HRP uses diluent to dilute in 1: 1000 ratio; Biotin-Tyramine uses diluent to dilute in 1: 500 ratio; Streptavidin-Nanogold uses diluent to dilute in 1: 40 ratio.
1. the Streptavidin-HRP 10 μ l after each hybridization region of chip adds dilution place 30min for 37 ℃; Take out the back and clean 20s with the PBST washing lotion, triplicate is put room temperature and is dried.2. the Biotin-Tyramine 10 μ l after each hybridization region of chip adds dilution place 30min for 37 ℃; Take out the back and clean 20s with the PBST washing lotion, triplicate is put room temperature and is dried.3. the Streptavidin-Nanogold 10 μ l after each hybridization region of chip adds dilution place 30min for 37 ℃; Take out the back and clean 20s with the PBST washing lotion, triplicate is used deionized water rinsing, puts room temperature and dries.4. silver-colored transfection reagent A liquid and B liquid equal-volume are mixed, A, the B mixed solution of each hybridization region lucifuge adding immediately 30ul are treated to occur on the chip naked eyes and are as seen stopped colour developing behind grey or the black round dot clearly, and developing time is about 2min30s, use washed with de-ionized water, dry.
The visual chip scanner that uses Shenzhen Pu Ruikang Bioisystech Co., Ltd to produce scans, and with ArrayVision7.0 software analysis result.
Embodiment 2: the determining of the positive criterion of gene chip
The Cutoff value is to judge whether positive standard of gene chip signal value, every typing probes is chosen non-Respirovirus (being negative strain) respectively, blank carries out gene chip hybridization, by experiment and data statistics repeatedly, with the background statistical average value+2SD of negative strain and the blank Cutoff value as every probe, see Table 6.With the separating capacity of each Respirovirus detection probes more than 2.5 times as the judging criterion of this virus-positive.
Determining of each probe Cutoff value of table 6
Embodiment 3: the visual detection gene chip of important respiratory tract Causative virus specificity is estimated
Specificity is the most important performance assessment criteria of diagnostic method, and the present invention uses and optimizes good system and condition, has detected 9 strains such as various common Respirovirus, and chip detection the results are shown in accompanying drawing 3.As seen from the figure, utilize the present invention 9 kinds of common Respiroviruses correctly can be distinguished, specificity is good.
Embodiment 4: the sensitivity of the visual detection gene chip of important respiratory tract Causative virus is estimated
In order to estimate the sensitivity of each detection probes of chip, we have made up the plasmid of each Respirovirus amplified target gene and in-vitro transcription RNA as with reference to product.Be template with various Respirovirus RNA, use corresponding primer (downstream primer is mark) to carry out single stage method RT-PCR, product is connected to PGM-T vector (TIANGEN) through using the T4DNA ligase enzyme behind the purifying, transformed competence colibacillus bacillus coli DH 5 alpha (TIANGEN), correct through sequence verification recombinant plasmid sequence.Plasmid DNA being cut with restriction enzyme Spe I enzyme carry out linearizing and reclaim purifying, is template with linearizing DNA, according to
The step of Large Scale RNA Production System-T7 test kit (Promega) specification sheets is carried out in-vitro transcription, transcription product is after the imitative extracting of phenol, isopropanol precipitating, with quantitatively stand-by after the water dissolution of no RNase enzyme, packing and in-70 ℃ of preservations, as in-vitro transcription RNA reference material.9 kinds of outer transcribe rna reference materials of virosome that contain target gene have been prepared altogether.Every kind of in-vitro transcription RNA reference material gradient dilution prepares the sensitivity reference material.
Use Respirovirus sensitivity reference material that the detection sensitivity of chip is estimated, found that chip all can detect 10 to every kind of Respirovirus
2The in-vitro transcription RNA of copies/ system, the detection of chip is limited to 10
2Copies/ system in-vitro transcription RNA.Be example with human metapneumovirus and acute laryngo-tracheo-bronchitis virus, select 10
5Copies/ μ l, 10
4Copies/ μ l, 10
3Copies/ μ l, 10
2Copies/ μ l, 10
1Chip detection is carried out in the in-vitro transcription RNA of copies/ μ l and positive and negative contrast, the results are shown in accompanying drawing 4.As seen from the figure, chip all can detect 10 to human metapneumovirus and acute laryngo-tracheo-bronchitis virus
2The in-vitro transcription RNA of copies/ system.
Respiratory syncytial virus, influenza virus B type have also used other mode to carry out the sensitivity evaluation, and virus-culturing fluid extracts RNA after through 10 times of gradient dilutions.Use and optimize good chip method detection respiratory syncytial virus, the RNA that the result extracts after respiratory syncytial virus is through 1000 times of dilutions, chip detection respiratory syncytial virus probe signals value still greater than corresponding cutoff value, namely can use the method for chip to detect.Use and optimize good chip method detection influenza virus B type, the RNA that the result extracts after virus is through 100000 times of dilutions, chip detection influenza B virus probe signals value namely can use the method for chip to detect still greater than corresponding Cutoff value.RT-PCR product electrophoresis result is seen accompanying drawing 5, and corresponding chip detection the results are shown in accompanying drawing 6, and the detection sensitivity of chip will be higher than electrophoretic method as seen from the figure.
In addition to the implementation, the present invention also has other embodiments.Every employing is equal to the technical scheme of replacement or equivalent transformation formation, all in the protection domain that the present invention requires.
Claims (1)
1. visual gene chip that detects common respiratory tract Causative virus nucleic acid, can be used for the detection of A, Type B influenza virus, parainfluenza virus 1,2 types, human metapneumovirus, respiratory syncytial virus, Measles virus, rubella virus, mumps virus, its preparation method comprises:
1) step 1:
Prepare 9 pairs of Respirovirus Auele Specific Primers, put into 3 pipe RT-PCR systems respectively, sequence is as shown in table 1:
Table 1 primer sequence
2) step 2:
Prepare 9 virus-specific probes, sequence is as shown in table 2:
Table 2 virus-specific probe sequence
3) step 3:
With each oligonucleotide probe in the step 2 with 6 * SSC, 0.1%SDS is diluted to final concentration 50 μ M, and point is to the aldehyde radical modification slide of blank, array is as shown in table 3, wherein sheet matrix control probe is the 20T sequence, 5 ' end cy3 mark, 3 ' is held amido modified, and the point sample amount of all probes is 3nl, places dry 18 hours in room temperature at least before the use;
Table 3 oligonucleotide probe array
4) step 4:
Preparation RT-PCR system, it is as shown in table 4 to fill a prescription:
Table 4RT-PCR system formulation
5) step 5:
The preparation hybridization solution, it consists of 9 * SSC, 0.3%SDS, 11% methane amide, 11%50 * Denhardt ' s; Preparation washing lotion A, it consists of 1 * SSC, 0.2%SDS; Preparation washing lotion B, it consists of 0.2 * SSC; Preparation washing lotion C, it consists of 0.1 * SSC;
6) step 6:
Prepare visual detection reagent, comprise Streptavidin-HRP, Biotin-Tyramine, Streptavidin-Nanogold, diluent, PBST washing lotion, silver-colored transfection reagent A liquid and B liquid, wherein the diluent component is 1 * PBS+0.1%BSA, PBST washing lotion component is 1 * PBS+0.05%Tween20, silver transfection reagent A fluid component is the aqueous solution of Silver monoacetate, concentration 4mg/mL, silver-colored transfection reagent B fluid component are the citrate buffer solution solution of quinhydrones, concentration 10mg/mL.
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| CN105087824B (en) * | 2015-03-04 | 2019-10-25 | 浙江省疾病预防控制中心 | The preparation and use of Hemorrhagic fever correlation pathogen sldh gene chip |
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| CN101613767A (en) * | 2009-07-30 | 2009-12-30 | 港龙生物科技有限公司 | The gene chip of the multiple respirovirus of parallel detection |
| CN101942524A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Combined nucleic acid real-time fluorescent detection method for influenza A H1N1 virus and influenza A virus and kit |
| CN101985665A (en) * | 2010-11-12 | 2011-03-16 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101942524A (en) * | 2009-07-08 | 2011-01-12 | 中国科学院广州生物医药与健康研究院 | Combined nucleic acid real-time fluorescent detection method for influenza A H1N1 virus and influenza A virus and kit |
| CN101613767A (en) * | 2009-07-30 | 2009-12-30 | 港龙生物科技有限公司 | The gene chip of the multiple respirovirus of parallel detection |
| CN101985665A (en) * | 2010-11-12 | 2011-03-16 | 复旦大学 | Method for detecting various respiratory viruses and primers and probes thereof |
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