CN100412205C - Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its prepn and application - Google Patents
Membrane gene chip for simultaneous detection of three groups, A, B and C, of human rotaviruses and its prepn and application Download PDFInfo
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- CN100412205C CN100412205C CNB2005101043382A CN200510104338A CN100412205C CN 100412205 C CN100412205 C CN 100412205C CN B2005101043382 A CNB2005101043382 A CN B2005101043382A CN 200510104338 A CN200510104338 A CN 200510104338A CN 100412205 C CN100412205 C CN 100412205C
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Abstract
The present invention discloses a membrane chip for simultaneously detecting human rotaviruses of groups A, B and C, which comprises a nylon membrane with positive charges, oligonucleotide probes, and a contrast and blank point coating, wherein the oligonucleotide probes and the contrast and blank point coating are distributed on a glass substrate in an array mode. The point coating comprises 6 specific human rotavirus detecting probes in different groups, one positive contrast probe, four negative contrast probes and a blank sample liquid contrast. Digoxin is added to mark the positive contrast probe 5' end. The present invention also discloses a preparation method of the chip, and the application of the chip using as a clinical diagnostic reagent. The chip of the present invention adopts a multiple PCR amplification mode to cause the nucleic acid which is to be detected to be marked with the digoxin, and hybridization results are obtained by immunological coloration. The present invention can be used for the gene detection and the group identification of a plurality of groups of human rotaviruses simultaneously, have wide application prospect, and probably satisfy the food sanitation detection and the need for detecting human rotaviruses in the field of disease prevention, disease control and clinical medical treatment.
Description
Technical field
The present invention relates to a kind of biochip and preparation method thereof and application of gene microarray, relate in particular to a kind of membrane gene chip and preparation method and application that detects A, B, three groups of human rotaviruss of C simultaneously, belong to biochip and diagnostic reagent technical field.
Background technology
Gene chip is meant many specific oligonucleotide fragments or gene fragment as probe, clocklike arrange and be fixed on the solid support, hybridize by the base pairing principle with the nucleic acid samples of mark to be measured then, through certain detection system hybridization signal is detected again, and be equipped with computer system each hybridization signal is carried out data analysis and processing, thereby draw desired information rapidly.Compare with traditional hybridization technique, this technology has microminiaturization, high sensitivity, the distinguishing feature of height collimation and high speed.Its tempo and the technology of wherein being used attract people's attention.Up to now, detect in genetic expression, detection in Gene Mutation, extensive studies and application have been launched in aspects such as single nucleotide polymorphism and determined dna sequence.
(Rotavirus RV) is the main cause of disease of whole world humans and animals acute diarrhea to rotavirus, endangers huge.By the antigenic specificity of its structural protein VP6, rotavirus is divided into A～G totally seven groups, and that can infect the mankind only has three groups of A, B, C.In developing country, rotavirus causes 87.3 10000 death of child below 5 years old every year, occupies second in children's cause of the death; Diarrhoea also occupies second in the Chinese children cause of the death, autumn and winter, children's diarrhae 50% was to be caused by rotavirus, directly threatens public safety, social stability, Economic development and the population quality of country.
For the detection of rotavirus, mainly be at virus antigen in the ight soil or gene at present, the technology of having set up can reduce following 3 classes:
1. virus is separated and morphologic observation: but the electron microscope that is used for morphologic observation costs an arm and a leg, and descend for the detection sensitivity of the virus of degrading, be not suitable for a large amount of samples, also be not suitable for basic unit and use.
2. immunological technique: the most at present using more, mainly comprise enzyme immunological technique [EIA, comprise enzyme linked immunosorbent assay (ELISA) and film immunity test (MEIA)], technology such as latex agglutination test (LA) and colloid gold test paper, all have commercial kit to provide abroad, domestic also not commercialization.It is higher that immunological technique has susceptibility, the better and simple and rapid characteristics of specificity.But test sample is very restricted, and solid thing to be checked may detect hardly in this way.
3. viral nucleic acid detects: mainly comprise methods such as polyacrylamide gel electrophoresis (PAGE) and RT-PCR.The former can directly detect rotavirus according to the nucleic acid banding pattern in the PAGE glue, but sensitivity is not high.Than immunological technique and PAGE glue technology, round pcr not only has higher sensitivity, and almost is applicable to all foodstuff samples.In addition, this method is not for providing a kind of simple and reliable alternative method with the sample that serological method carries out somatotype.But the susceptibility height of amplification has brought the false positive problem simultaneously, then can't not verify the exactness of amplified production by hybridization.
In addition, also there is a common shortcoming in above-mentioned detection method: flux is low, i.e. one-time detection reaction only can detect the rotavirus of a kind of group or type, and powerless for once detecting all many group rotavirus that can cause the people to infect.
By retrieval, the gene chip about the PCR product that utilizes digoxigenin labeled and membrane gene chip hybridization detect many group rotavirus does not simultaneously appear in the newspapers so far.
Summary of the invention
At the deficiencies in the prior art, one of the technical problem to be solved in the present invention provides a kind of membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously, with three group rotavirus and the low defective of detection sensitivity that can not one-time detection cause the people to suffer from diarrhoea that overcomes that prior art exists, satisfy monitoring for food hygiene, the needs in prevention and control of diseases and clinical treatment field;
Two of the technical problem to be solved in the present invention is Gaoxin labeling techniques in combination, is substrate with positively charged nylon membrane, provides a kind of to the described membrane gene chip production method that detects A, B, three groups of human rotaviruss of C simultaneously;
Three of the technical problem to be solved in the present invention is to disclose the described application of membrane gene chip in the preparation reagent for clinical diagnosis that detects A, B, three groups of human rotaviruss of C simultaneously.
Detect A, B, three groups of human rotaviruss' of C membrane gene chip when the present invention relates to, comprise oligonucleotide probe and contrast and barren point coating that nylon membrane substrate and array distribute, it is characterized in that described nylon membrane substrate is positively charged nylon membrane; Described oligonucleotide probe and contrast and barren are selected coating and are distributed on the nylon membrane substrate, comprise human rotavirus's group-specific detection probes of 6 different groups, 1 positive control probe, and 4 negative control probes and the contrast of blank sampling liquid, wherein,
The gene order of described 6 human rotavirus's group-specific detection probes is:
ProbeA1：5′-ATTTATTGAATGCTTCGATAT-3′
ProbeA2：5′-ACTGGTGAGTGGATTGTTTGA-3′
ProbeB1：5′-ATCCCATTTGAGTAAATTCAG-3′
ProbeB2：5′-ATGATAATTCAGCCAAGCC-3′
ProbeC1：5′-CTGTATTAGCTACATGACCGT-3′
ProbeC2：5′-AGCTATTGGAGTTTGGTAGTT-3′
The gene order of described 1 positive control probe is:
PbP：5′-ATTTATTGAATGCTTCGATAT-3′
The gene order of described 4 negative control probes is respectively:
PbEC：5′-GATGAGAATGTGCCTTCGGGAA-3′
PbSA：5′-GAACATATGTGTAAGTAACTGTGC-3′
PbHBV：5′-AGAAAGACCTTTAACCTA-3′
PbPSE：5′-TAGGTGGTTCAGCAAGTTGGAT-3′
Above-mentioned positive control probe is at its 5 ' end band digoxigenin labeled;
Above-mentioned every kind of probe is made 2 parallel points with contrast and barren point coating at nylon membrane substrate surface point, and the diameter of each point is 0.25mm, and dot spacing is 0.8mm, and the size of some coating microarray is 4.5mm * 5.5mm.
Detect in A, B, three groups of human rotaviruss' of C the membrane gene chip in above-mentioned, described positive control probe distribution is positioned at the upper left corner of coating array and the lower left corner.
The membrane gene chip production method that detects A, B, three groups of human rotaviruss of C simultaneously of the present invention, its step is as follows:
(1) determine probe: according to A, B, three groups of human rotavirus's gene orders of C, select to have determined 6 human rotavirus's group-specific detection probes that its gene order is:
ProbeA1：5′-ATTTATTGAATGCTTCGATAT-3′
ProbeA2：5′-ACTGGTGAGTGGATTGTTTGA-3′
ProbeB1：5′-ATCCCATTTGAGTAAATTCAG-3′
ProbeB2：5′-ATGATAATTCAGCCAAGCC-3′
ProbeC1：5′-CTGTATTAGCTACATGACCGT-3′
ProbeC2：5′-AGCTATTGGAGTTTGGTAGTT-3′
PbP：5′-ATTTATTGAATGCTTCGATAT-3′
PbEC：5′-GATGAGAATGTGCCTTCGGGAA-3′
PbSA：5′-GAACATATGTGTAAGTAACTGTGC-3′
PbHBV：5′-AGAAAGACCTTTAACCTA-3′
PbPSE：5′-TAGGTGGTTCAGCAAGTTGGAT-3′
(2) chip preparation: with human rotavirus's group-specific detection probes of above-mentioned selection, positive control probe and negative control probe, above-mentioned probe being diluted respectively as diluent with 3 * SSC is 10-100 μ M, with the sampling liquid is blank, being equipped with SMP3 point needle contact with biochip point sample instrument Pixsys5500 selects on positively charged nylon membrane substrate, liquid level is 4-6mm in the sampling plate, pin residence time in thief hole is 20ms, during point sample, pin is 10ms in the on-chip residence time of nylon membrane, every kind of probe is made 2 parallel points in 1 * 2 mode at nylon membrane substrate surface point with contrast and barren point coating, wherein, the positive control probe is with digoxigenin labeled at its 5 ' end, and described positive control probe distribution is positioned at the upper left corner of coating array and the lower left corner, the diameter of each point is 0.25mm, and dot spacing is 0.8mm, and the size of some coating microarray is 4.5mm * 5.5mm; Chip after the system is lain against in the UV-crosslinked instrument 4500J/cm
2Crosslinked 3min promptly gets the described membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously down.
In above-mentioned, detect in A, B, three groups of human rotaviruss' of C the membrane gene chip production method, the selection principle of described human rotavirus's group-specific detection probes is: according to A, B, three groups of human rotavirus's gene orders of C, design is at every group of human rotavirus's specific oligonucleotide probe, make the melting temperature(Tm) of every probe close, and the melting temperature(Tm) of every probe is in ± 3 ℃, to avoid the formation of hair clip and dimeric structure.
In above-mentioned, detect in A, B, three groups of human rotaviruss' of C the membrane gene chip production method, described positive control probe its 5 ' end with on be digoxin with the ordinary method mark.
The application of membrane gene chip in the preparation reagent for clinical diagnosis that detects A, B, three groups of human rotaviruss of C simultaneously of the present invention.Utilize chip of the present invention to organize the human rotavirus to A, B organizes the human rotavirus, and C organizes the human rotavirus or simultaneously A, B, three groups of human rotaviruss of C detected; For example: use the membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously of the present invention clinically and can detect and make quick diagnosis to the infantile diarrhea sample that doubtful A group RV infects.
In above-mentioned, detect in A, B, three groups of human rotaviruss' of C the application of membrane gene chip in the preparation reagent for clinical diagnosis,
Sample prepares as follows:
Extract rotavirus gene group RNA with Trizol reagent (U.S. OMEGA company) by operation instruction, and dissolve RNA with 0.5 ‰ DEPC (tetra-sodium diethyl ester) that the autoclaving of application quantity is crossed;
The sequence of each the group-specific upstream and downstream primer that designs respectively at A, B, three groups of human rotaviruss of C is:
A organizes the human rotavirus:
Upstream primer: 5 '-AACGAACAGTATGTTTCATCA-3 '
Downstream primer: 5 '-TTGAAGGTCGTGATTGTGTTG-3 '
B organizes the human rotavirus:
Upstream primer: 5 '-TGGAACATACGAAGTTACAG-3 '
Downstream primer: 5 '-TACATCATAGTCTCGATAAAATA-3 '
C organizes the human rotavirus:
Upstream sequence: 5 '-GAAGAGTAGCATCAGGGAC-3 '
Downstream primer: 5 '-TAAACGTAAATCATTGATTT-3 '
Above-mentioned primer is characterised in that melting temperature(Tm) is 53-55 ℃, and GC% is 40-60%, and has avoided hairpin structure and dimeric structure.
Detect in above-mentioned in A, B, three groups of human rotaviruss' of C the application of membrane gene chip in the preparation reagent for clinical diagnosis, the marker that is used for labelling human rotavirus group-specific upstream primer is a digoxin.
With the above-mentioned upstream primer of digoxigenin labeled, contain at the same time in the pcr amplification system of above-mentioned A, B, three groups of human rotavirus's upstream and downstream of C primer, in multiple RT-PCR amplification mode, amplification when realization is organized the human rotavirus or organized the human rotavirus more list.
RNA is carried out RNA PCR (AMV) the VER 2.1 test kit specification sheetss operation that reverse transcription reaction is produced according to Takara company.Set up the PCR system then: contain in the 25 μ l reaction systems: 1.5 μ l MgCl
2, 2.5 μ l, 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 16.4 μ l sterilizes, 0.125 μ l Taq enzyme (5U/ μ l), 1.0 μ l primer mixtures (the A group human rotavirus's of digoxigenin labeled upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), 0.5 μ l template.
The PCR program is: 94 ℃ of pre-sex change 2min, and then by 94 ℃ of 45sec, 52 ℃ of 45sec, 72 ℃ of 1min are a circulation, after 30 circulations, 72 ℃ are extended 5min again.
Pcr amplification product and many group human rotaviruss detect the hybridization of gene chip:
Get an amount of amplified production and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds an amount of hybridization mixed solution, hybridizes 10-240min under 48-55 ℃ hybridization temperature.
Immunity colour developing and result read:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 30-40 ℃ of reaction 10-30min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 30-40 ℃ of colour developing down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
The membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously of the present invention has following advantage and effect:
(1) upstream primer of digoxigenin labeled of the present invention is realized amplification and mark to viral nucleic acid to be checked, and the nylon membrane of combined belt positive charge, can not pass through specific instrument, and with the naked eyes direct viewing and judge experimental result, be more suitable for using in grass-roots unit.
(2) of the present invention with the substrate of positively charged nylon membrane as gene chip, can use with ordinary method synthetic oligonucleotide as probe, need save cost greatly than adopting other material with chemical group and connecting arm modified oligonucleotide as substrate.
(3) of the present invention with the substrate of positively charged nylon membrane as gene chip, owing to nylon membrane is a kind of porous material and has water permeability,, can fix a large amount of nucleic acid molecule, thereby improve the sensitivity that detects as the carrier that sticks nucleic acid.
(4) many group human rotaviruss of the present invention detect gene chip and use the multiplex PCR amplification, the upstream and downstream primer that promptly in the pcr amplification system, adds three groups of human rotaviruss simultaneously, can realize detection, augmentation detection when also can realize to two groups and three groups of human rotaviruss to lineup's rotavirus.
(5) human rotavirus's group-specific detection probes of the present invention can realize human rotavirus's detection and grouping are identified.
(6) of the present invention with all specific probe points of three groups of human rotavirus A, B, C that can cause human diarrhoea on same detection microarray, detect when can realize these three groups of human rotaviruss.
In a word, the chip that the present invention relates to adopts multiplex PCR amplification mode, can be used for simultaneously many groups human rotavirus's gene test and group are identified, has broad application prospects, be expected to satisfy monitoring for food hygiene, the needs that prevention and control of diseases and clinical treatment field are detected the human rotavirus.
Description of drawings
Fig. 1: the probe layout viewing that detects A, B, three groups of human rotaviruss' of C membrane gene chip simultaneously of the present invention,
Wherein: described probe arrange into:
a1，a2，：DIG-PbP(10μmol/L)；a3，a4：3×SSC；a5，a6，：3×SSC；
b1，b2：PbA1(10μmol/L)；b3，b4：PbA2(10μmol/L)；b5，b6：3×SSC；
c1，c2：PbB1(10μmol/L)；c3，c4：PbB2(10μmol/L)；c5，c6：3×SSC；
d1，d2：PbC1(10μmol/L)；d3，d4：PbC2(10μmol/L)；d5，d6：3×SSC；
e1，e2：PbEC(10μmol/L)；e3，e4：PbSA(10μmol/L)；e5，e6：3×SSC；
f1，f2：DIG-PbP(10μmol/L)；f3，f4：PbHBV(10μmol/L)；f5，f6：PbPSE(10μmol/L)。
Fig. 2: agarose gel electrophoresis detects pcr amplification product
Wherein: described each swimming lane is:
Swimming lane M is nucleic acid molecule marker DL2000; Swimming lane 1 is a pcr amplification of described in the embodiment 2 A being organized the human rotavirus; Swimming lane 2 is a pcr amplification of described in the embodiment 3 B being organized the human rotavirus; Swimming lane 3 is a pcr amplification of described in the embodiment 4 C being organized the human rotavirus; Swimming lane 4 is to A, B, three groups of human rotaviruss' of C pcr amplification described in the embodiment 5.
The hybridization scanning result of the homogenic chip of Fig. 3: A group human rotavirus's pcr amplification product
The result shows that A group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects A group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects B, C group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
The hybridization scanning result of the homogenic chip of Fig. 4: B group human rotavirus's pcr amplification product
The result shows that B group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects B group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects A, C group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
The hybridization scanning result of the homogenic chip of Fig. 5: C group human rotavirus's pcr amplification product
The result shows that C group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects C group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects A, B group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
The hybridization scanning result of Fig. 6: A, B, three groups of homogenic chips of human rotavirus's pcr amplification product of C
The result shows that A, B, three groups of human rotavirus's pcr amplification products of C positive findings all occurs after hybridizing with the specific probe that detects A, B, three groups of human rotaviruss of C on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
Embodiment
Below in conjunction with embodiment the content of invention is further elaborated:
(1) determine probe: according to A, B, three groups of human rotavirus's gene orders of C, select to have determined 6 human rotavirus's group-specific detection probes that its gene order is:
ProbeA1：5′-ATTTATTGAATGCTTCGATAT-3′
ProbeA2：5′-ACTGGTGAGTGGATTGTTTGA-3′
ProbeB1：5′-ATCCCATTTGAGTAAATTCAG-3′
ProbeB2：5′-ATGATAATTCAGCCAAGCC-3′
ProbeC1：5′-CTGTATTAGCTACATGACCGT-3′
ProbeC2：5′-AGCTATTGGAGTTTGGTAGTT-3′
PbP：5′-ATTTATTGAATGCTTCGATAT-3′
PbEC：5′-GATGAGAATGTGCCTTCGGGAA-3′
PbSA：5′-GAACATATGTGTAAGTAACTGTGC-3′
PbHBV：5′-AGAAAGACCTTTAACCTA-3′
PbPSE：5′-TAGGTGGTTCAGCAAGTTGGAT-3′
(2) chip preparation: above-mentioned probe being diluted respectively as diluent with 3 * SSC is 10 μ M, with 3 * SSC is blank, with 5 '-DIG-PbA1-3 ' (is designated as: DIG-PbP) as positive control, (be designated as: PbEC) with the intestinal bacteria specific probe, staphylococcus aureus specific probe: (be designated as: PbSA), HBV hepatitis B virus specific probe (is designated as: PbHBV), the Pseudomonas aeruginosa specific probe (is designated as: PbPSE) as negative control, being equipped with SMP3 point needle contact with biochip point sample instrument Pixsys5500 selects on described positively charged nylon membrane substrate, liquid level is 4-6mm in the sampling plate, pin residence time in thief hole is 20ms, during point sample, pin is 10ms in the on-chip residence time of nylon membrane, every kind of probe is made 2 parallel points in 1 * 2 mode at nylon membrane substrate surface point with contrast and barren point coating, wherein, the positive control probe is with digoxigenin labeled with ordinary method at its 5 ' end, and described positive control probe distribution is positioned at the upper left corner of coating array and the lower left corner, the diameter of each point is 0.25mm, dot spacing is 0.8mm, and the size of some coating microarray is 4.5mm * 5.5mm; Chip after the system is lain against in the UV-crosslinked instrument 4500J/cm
2Crosslinked 3min promptly gets the described membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously down.
(1) people A group human rotavirus standard strain RV5 and Wa strain are carried out cell cultures, extract the viral RNA in the nutrient solution supernatant, RNA is carried out reverse transcription reaction with the operation of the RNA PCR of Takara company (AMV) VER 2.1 test kit specification sheetss.Set up the PCR system then: contain in the 25 μ l reaction systems: 1.5 μ l MgCl
2, 2.5 μ l, 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 16.4 μ l sterilizes, 0.125 μ lTaq enzyme (5U/ μ l), 1.0 μ l primer mixtures (, the A of digoxigenin labeled group human rotavirus's upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), 0.5 μ lRT product.
(2) electrophoresis detection of pcr amplification product
Get PCR product 5 μ l, carry out observations behind the electrophoresis, see Fig. 2 with 1% sepharose that contains ethidium bromide.
(3) pcr amplification product is with the membrane gene chip hybridization that detects many group human rotaviruss
Get 50 μ l amplified productions and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds 60 μ l hybridization mixed solution, hybridizes 30min under 55 ℃ hybridization temperature.
(4) reading of immunity colour developing and result:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 37 ℃ of reaction 15min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 37 ℃ of colour developings down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage (see figure 3) with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
The result shows that A group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects A group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects B, C group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
(1) people B group human rotavirus target nucleic acid sequence is set up the pcr amplification system
Contain in the 25 μ l reaction systems: 1.5 μ l MgCl
2, 2.5 μ l, 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 16.4 μ l sterilizes, 0.125 μ lTaq enzyme (5U/ μ l), 1.0 μ l primer mixtures (the A group human rotavirus's of digoxigenin labeled upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), 0.5 μ l template.
The PCR program is: 94 ℃ of pre-sex change 2min of C, and then by 94 ℃ of 45sec, 52 ℃ of 45sec, 72 ℃ of 1min are a circulation, after 30 circulations, 72 ℃ are extended 5min again.
(2) electrophoresis detection of pcr amplification product
Get PCR product 5 μ l, carry out observations behind the electrophoresis, see Fig. 2 with 1% sepharose that contains ethidium bromide.
(3) pcr amplification product is with the membrane gene chip hybridization that detects many group human rotaviruss
Get 50 μ l amplified productions and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds 60 μ l hybridization mixed solution, hybridizes 30min under 55 ℃ hybridization temperature.
(4) reading of immunity colour developing and result:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 37 ℃ of reaction 15min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 37 ℃ of colour developings down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage (see figure 4) with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
The result shows that B group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects B group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects A, C group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
(1) people C group human rotavirus target nucleic acid sequence is set up the pcr amplification system
Contain in the 25 μ l reaction systems: 1.5 μ l MgCl
2, 2. μ ll 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 16.4 μ l sterilizes, 0.125 μ lTaq enzyme (5U/ μ l), 1.0 μ l primer mixtures (the A group human rotavirus's of digoxigenin labeled upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), 0.5 μ l template.
The PCR program is: 94 ℃ of pre-sex change 2min, and then by 94 ℃ of 45sec, 52 ℃ of 45sec, 72 ℃ of 1min are a circulation, after 30 circulations, 72 ℃ are extended 5min again.
(2) electrophoresis detection pcr amplification product
Get PCR product 5 μ l, carry out observations behind the electrophoresis, see Fig. 2 with 1% sepharose that contains ethidium bromide.
(3) pcr amplification product is with the membrane gene chip hybridization that detects many group human rotaviruss
Get 50 μ l amplified productions and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds 60 μ l hybridization mixed solution, hybridizes 30min under 55 ℃ hybridization temperature.
(4) reading of immunity colour developing and result:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 37 ℃ of reaction 15min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 37 ℃ of colour developings down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage (see figure 5) with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
The result shows that C group human rotavirus pcr amplification product positive findings occurs after hybridizing with the specific probe that detects C group human rotavirus on the chip, negative findings then occurs with the specific probe hybridization that detects A, B group human rotavirus on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
(1) A, B, three groups of human rotavirus's target nucleic acid sequences of C are set up the multiplex PCR amplification system
25 μ l; Contain in the reaction system: 1.5 μ l MgCl
2, 2.5 μ l, 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 15.4 μ l sterilizes, 0.125 μ lTaq enzyme (5U/ μ l), 1.0 μ l primer mixtures (the A group human rotavirus's of digoxigenin labeled upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), three groups of each 0.5 μ l of template.
The PCR program is: 94 ℃ of pre-sex change 2min, and then by 94 ℃ of 45sec, 52 ℃ of 45sec, 72 ℃ of 1min are a circulation, after 30 circulations, 72 ℃ are extended 5min again.
(2) three groups of human rotaviruss' of electrophoresis detection multiplex PCR amplified production
Get PCR product 5 μ l, carry out observations behind the electrophoresis, see Fig. 2 with 1% sepharose that contains ethidium bromide.
(3) pcr amplification product is with the membrane gene chip hybridization that detects many group human rotaviruss
Get 50 μ l amplified productions and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds 60 μ l hybridization mixed solution, hybridizes 30min under 55 ℃ hybridization temperature.
(4) reading of immunity colour developing and result:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 37 ℃ of reaction 15min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 37 ℃ of colour developings down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage (see figure 6) with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
The result shows that A, B, three groups of human rotavirus's pcr amplification products of C positive findings all occurs after hybridizing with the specific probe that detects A, B, three groups of human rotaviruss of C on the chip.Also occur positive signal on the position of positive control point, all occurred negative findings on the position of negative control and blank point.
The infantile diarrhea sample that embodiment 6 utilizes the clinical doubtful A group RV of membrane gene chip detection 92 examples that detects A, B, three groups of human rotaviruss of C simultaneously of the present invention to infect
(1) extraction of RNA in the infantile diarrhea sample
With 1 * PBS fecal sample is diluted 10 times, the centrifugal 2min of 10000rpm; Get 300 μ l supernatants, add 30 μ l 10%SDS, 56 ℃ of insulation 30min; Add 800 μ l Trizol reagent, room temperature is placed 5min; Add 200 μ l chloroforms, behind the vortex concussion 15sec, room temperature leaves standstill 10min; 4 ℃ centrifugal, the centrifugal 10min of 12000rpm; The careful upper strata liquid of drawing adds 500 μ l Virahols to the 1.5ml centrifuge tube, place 10min under the room temperature; 4 ℃ centrifugal, the centrifugal 10min of 12000rpm; The careful suction abandoned supernatant, adds 1ml 75% ethanol (the 0.5 ‰ DEPC water preparation behind the autoclaving), and 4 ℃ centrifugal, the centrifugal 5min of 7500rpm; Abandon ethanol, add 20 μ l, 0.5 ‰ DEPC water (autoclaving) dissolving after the drying ,-80 ℃ of storages.
(2) pcr amplification
With the operation of the RNA PCR of Takara company (AMV) VER 2.1 test kit specification sheetss RNA is carried out reverse transcription reaction.Set up the PCR system then: contain in the 25 μ l reaction systems: 1.5 μ l MgCl
2, 2.5 μ l, 10 * PCR damping fluid (does not contain Mg
-2), 2.0 μ ldNTP (2.5mmol/L), the distilled water 15.4 μ l sterilizes, 0.125 μ lTaq enzyme (5U/ μ l), 1.0 μ l primer mixtures (the A group human rotavirus's of digoxigenin labeled upstream primer, A group human rotavirus's downstream primer, the B group human rotavirus's of digoxigenin labeled upstream primer, B group human rotavirus's downstream primer, the C group human rotavirus's of digoxigenin labeled upstream primer, each 10 μ mol/L of downstream primer of C group human rotavirus), 0.5 μ l RT product.
The PCR program is: 94 ℃ of pre-sex change 2min, and then by 94 ℃ of 45sec, 52 ℃ of 45sec, 72 ℃ of 1min are a circulation, after 30 circulations, 72 ℃ are extended 5min again.
(3) electrophoresis detection of pcr amplification product
Get PCR product 5 μ l, carry out observations behind the electrophoresis with 1% sepharose that contains ethidium bromide.
(4) pcr amplification product is with the membrane gene chip hybridization that detects many group human rotaviruss
Get 50 μ l amplified productions and mix with the hybridization solution equal-volume, hybridization solution is 10 * Denhardt ' s, 10 * SSPE, 50 μ g/ml dextran, 0.1%SDS.To hybridize the mixed solution sex change: 100 ℃ of 10min.Place ice-water bath then rapidly.The film chip places the self-control plastics bag of suitable dimension, adds 60 μ l hybridization mixed solution, hybridizes 30min under 55 ℃ hybridization temperature.
(5) reading of immunity colour developing and result:
Hybridization is washed film 3min with distilled water after finishing; Wash film 10min with 5 * SSC/0.5%SDS then, immerse confining liquid (1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) sealing 30min.And then immerse in the antibody-solutions (0.02%Anti-DIG-AP, 1.0% sarracine, 0.1mol/L toxilic acid, 0.15mol/L NaCl) 37 ℃ of reaction 20min.Behind elutriant wash-out 5min, balance 2min in detecting diluent adds 2%NBT/BCIP again, 37 ℃ of colour developings down.When the color depth for the treatment of positive control reaches requirement,, the hybridization signal on the film chip is scanned storage with image with the AlphaScanTM scanner with distilled water flushing membrane chip termination reaction.
Detect A, B, three groups of human rotaviruss' of C membrane gene chip hybridization when the invention described above is set up after, detected result shows: A group human rotavirus detects male 83 examples, and negative have 9 examples.
(6) the infantile diarrhea sample that simultaneously the clinical doubtful A group RV of 92 identical examples is infected carries out ELISA test kit (Shenzhen peace all living creatures thing engineering corporation, product batch number: 20050115) Detection of antigen.
Detected result shows: 78 examples are the ELISA test kit Detection of antigen positive, and 14 examples are negative.
Prove that detection that we set up organizes the detection sensitivity of human rotavirus's membrane gene chip more and be higher than the wheel virus antigen detection kit of generally using clinically now.
Claims (8)
1. a membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously comprises oligonucleotide probe and contrast and barren point coating that nylon membrane substrate and array distribute, it is characterized in that described nylon membrane substrate is positively charged nylon membrane; Described oligonucleotide probe and contrast and barren are selected coating and are distributed on the nylon membrane substrate, comprise human rotavirus's group-specific detection probes of 6 different groups, 1 positive control probe, and 4 negative control probes and the contrast of blank sampling liquid, wherein,
The gene order of described 6 human rotavirus's group-specific detection probes is:
ProbeA1：5′-ATTTATTGAATGCTTCGATAT-3′
ProbeA2：5′-ACTGGTGAGTGGATTGTTTGA-3′
ProbeB1：5′-ATCCCATTTGAGTAAATTCAG-3′
ProbeB2：5′-ATGATAATTCAGCCAAGCC-3′
ProbeC1：5′-CTGTATTAGCTACATGACCGT-3′
ProbeC2：5′-AGCTATTGGAGTTTGGTAGTT-3′
The gene order of described 1 positive control probe is:
PbP：5′-ATTTATTGAATGCTTCGATAT-3′
The gene order of described 4 negative control probes is respectively:
PbEC：5′-GATGAGAATGTGCCTTCGGGAA-3′
PbSA：5′-GAACATATGTGTAAGTAACTGTGC-3′
PbHBV：5′-AGAAAGACCTTTAACCTA-3′
PbPSE：5′-TAGGTGGTTCAGCAAGTTGGAT-3′
Above-mentioned positive control probe is at its 5 ' end band digoxigenin labeled;
Above-mentioned every kind of probe is made 2 parallel points with contrast and barren point coating at nylon membrane substrate surface point, and the diameter of each point is 0.25mm, and dot spacing is 0.8mm, and the size of some coating microarray is 4.5mm * 5.5mm.
2. the membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously as claimed in claim 1 is characterized in that described positive control probe distribution is positioned at the upper left corner of coating array and the lower left corner.
3. the described membrane gene chip production method that detects A, B, three groups of human rotaviruss of C simultaneously of claim 1, its step is as follows:
(1) determine probe: according to A, B, three groups of human rotavirus's gene orders of C, select to have determined 6 human rotavirus's group-specific detection probes that its gene order is:
ProbeA1：5′-ATTTATTGAATGCTTCGATAT-3′
ProbeA2：5′-ACTGGTGAGTGGATTGTTTGA-3′
ProbeB1：5′-ATCCCATTTGAGTAAATTCAG-3′
ProbeB2：5′-ATGATAATTCAGCCAAGCC-3′
ProbeC1：5′-CTGTATTAGCTACATGACCGT-3′
ProbeC2：5′-AGCTATTGGAGTTTGGTAGTT-3′
Article 1, positive control probe, its gene order is:
PbP：5′-ATTTATTGAATGCTTCGATAT-3′
Article 4, negative control probe, its gene order is respectively:
PbEC：5′-GATGAGAATGTGCCTTCGGGAA-3′
PbSA：5′-GAACATATGTGTAAGTAACTGTGC-3′
PbHBV：5′-AGAAAGACCTTTAACCTA-3′
PbPSE：5′-TAGGTGGTTCAGCAAGTTGGAT-3′
(2) chip preparation: with human rotavirus's group-specific detection probes of above-mentioned selection, positive control probe and negative control probe, above-mentioned probe being diluted respectively as diluent with 3 * SSC is 10-100 μ M, with the sampling liquid is blank, being equipped with SMP3 point needle contact with biochip point sample instrument Pixsys5500 selects on positively charged nylon membrane substrate, liquid level is 4-6mm in the sampling plate, pin residence time in thief hole is 20ms, during point sample, pin is 10ms in the on-chip residence time of nylon membrane, every kind of probe is made 2 parallel points in 1 * 2 mode at nylon membrane substrate surface point with contrast and barren point coating, wherein, the positive control probe is with digoxigenin labeled at its 5 ' end, and described positive control probe distribution is positioned at the upper left corner of coating array and the lower left corner, the diameter of each point is 0.25mm, and dot spacing is 0.8mm, and the size of some coating microarray is 4.5mm * 5.5mm; Chip after the system is lain against in the UV-crosslinked instrument 4500J/cm
2Crosslinked 3min promptly gets the described membrane gene chip that detects A, B, three groups of human rotaviruss of C simultaneously down.
4. the membrane gene chip production method that detects A, B, three groups of human rotaviruss of C simultaneously as claimed in claim 3, it is characterized in that, the selection principle of described human rotavirus's group-specific detection probes is: the difference of the melting temperature(Tm) of every probe is at ± 3 ℃, to avoid the formation of hair clip and dimeric structure.
5. the membrane gene chip production method that detects A, B, three groups of human rotaviruss of C simultaneously as claimed in claim 3 is characterized in that, described positive control probe its 5 ' end with on be digoxin with the ordinary method mark.
6. the described application of membrane gene chip in the preparation reagent for clinical diagnosis that detects A, B, three groups of human rotaviruss of C simultaneously of claim 1.
7. the application of membrane gene chip in the preparation reagent for clinical diagnosis that detects A, B, three groups of human rotaviruss of C simultaneously as claimed in claim 6, it is characterized in that the sequence of each the group-specific upstream and downstream primer that designs respectively at A, B, three groups of human rotaviruss of C is:
A organizes the human rotavirus:
Upstream primer: 5 '-AACGAACAGTATGTTTCATCA-3 '
Downstream primer: 5 '-TTGAAGGTCGTGATTGTGTTG-3 '
B organizes the human rotavirus:
Upstream primer: 5 '-TGGAACATACGAAGTTACAG-3 '
Downstream primer: 5 '-TACATCATAGTCTCGATAAAATA-3 '
C organizes the human rotavirus:
Upstream sequence: 5 '-GAAGAGTAGCATCAGGGAC-3 '
Downstream primer: 5 '-TAAACGTAAATCATTGATTT-3 '
8. as claim 6 or the 7 described application of membrane gene chip in the preparation reagent for clinical diagnosis that detect A, B, three groups of human rotaviruss of C simultaneously, it is characterized in that the marker that is used for labelling human rotavirus group-specific upstream primer in the PCR reaction is a digoxin.
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| CN100434535C (en) * | 2007-05-24 | 2008-11-19 | 清华大学 | Method of quantificationally detecting rota virus and special-purpose standard substance for the same |
| CN103224998A (en) * | 2013-04-24 | 2013-07-31 | 深圳市生科源技术有限公司 | Rotavirus PCR detection kit and detection method thereof |
| CN103757128B (en) * | 2014-02-05 | 2014-12-10 | 黄广平 | Paddy rice transgenic detection kit |
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