CN1508264A - Clinical infectious disease gene chip diagnosis scheme - Google Patents
Clinical infectious disease gene chip diagnosis scheme Download PDFInfo
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- CN1508264A CN1508264A CNA02128069XA CN02128069A CN1508264A CN 1508264 A CN1508264 A CN 1508264A CN A02128069X A CNA02128069X A CN A02128069XA CN 02128069 A CN02128069 A CN 02128069A CN 1508264 A CN1508264 A CN 1508264A
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Abstract
The invention relates to a clinic infectious disease gene chip diagnostic project: 1. make multiple PCR amplification on clinic sample DNA by peculiar PCR primers, then use the PCR primers to make multiple PCR amplification labeling, and make cross detection on the labeled product and the chip; 2. set blank comparison, able to eliminate the system pollution according to the blank comparison signal, and at the same time add UNG enzyme to effectively prevent the pollution of the previous PCR amplified product; 3. configure proper amplification circulating number to make the strength of the chip cross signal and the copy number of the template directly relative, thus able to make semi-quantitative determination.
Description
Technical field:
The present invention relates to a kind of clinical infectious disease gene chip diagnosis scheme, belong to biological technical field.
Background technology:
Biochip technology is a kind of emerging biotechnology, and it has wide practical use in each field of biology.Clinical diagnosis is one of its Application Areas.Infectious diseases is one of gene chip field of using the earliest, but the difficult point that the application clinically of this technology relates to is the contamination preventing of sensitivity, specificity, cost and pcr amplification.Common gene chip for clinical diagnosis detection scheme is: extract clinical sample DNA, use random primer labelling then, fluorochrome label in sample DNA, is hybridized detection with the DNA and the chip of mark.In this detection scheme, for once hybridize, do not have the amplification program of special primer, so specificity has only the hybridization program to guarantee, susceptibility can only rely on the high sensitivity decision of fluoroscopic examination.In order to improve detection sensitivity, have to increase labeled substrate, because costing an arm and a leg of labeled substrate causes cost too high, the human consumer is difficult to bear, thereby gene chip popularization and application clinically is restricted.
Summary of the invention:
The invention reside in set up one the cover be used for clinical infectious disease gene chip diagnosis scheme, the principle of work of the technical program is that this scheme is with laggard row labels of target molecule enrichment and hybridization to be detected:
1. clinical sample DNA is carried out multiplex PCR (multiplex PCR) amplification with the specific PCR primer, carry out the multiplex PCR amplification label with the specific PCR primer then, marked product is hybridized detection with chip again.This scheme combines the high specific of PCR high sensitivity and molecular hybridization, and marker mark target site sequence to be detected, has obtained cover high sensitivity, high specific and detection method cheaply.
2. this programme is provided with blank, can pollute by blank signal eliminating system, adds the UNG enzyme simultaneously to prevent the pollution of previous pcr amplification product effectively.In sample detection, make a pcr amplification blank, and use the marker mark different with the sample detection mark, hybridize detection with chip then with after the sample marker balanced mix, the ratio of the hybridization signal of the hybridization signal of marker and blank (ration) determines whether sample has positive signal per sample.
3. the positive template gradient is set, determines the pcr amplification cycle number, make the intensity of detection signal become positive correlation, thereby can carry out sxemiquantitative with the copy number of template.
The technical program compared with prior art, have specificity and highly sensitive, cost is low, can prevent the PCR product pollution effectively, detect advantages such as can carrying out sxemiquantitative.The technical program both had been applicable to clinical infectious disease gene chip diagnosis, was applicable to that also utilizing biochip technology to carry out all is population, monoid and individual evaluation of detecting target spot with the distinguished sequence.
Embodiment:
Enforcement of the present invention is undertaken by following program:
1. adopt existing DNA extraction method (having multiple fast and simply DNA extraction method to use at present), obtain sample DNA (or cDNA).
2. design of primers: according to the dna probe that is fixed on the chip, seek corresponding sequence, according to this section sequences Design PCR primer.Primer sequence is positioned at the then outer of probe sequence.Each dna probe designs a pair of primer, owing to will carry out the multiplex PCR amplification, the amplification condition of primer is wanted unanimity.This programme adopts Primer3 software to carry out design of primers under the default parameters condition.
3. multiplex PCR amplification, the pairing detection primer of all probes is mixed, the consumption of every kind of primer is (0.08-1pmol/ul) suitably, pcr amplification is that 20-25 circulation (is determined by experiment, make amplification efficiency less than platform effect), add UNG enzyme and dUTP in the amplified reaction system to prevent the PCR product pollution, all the other compositions of amplified reaction system and thermal circulation parameters scheme setting routinely, and consideration primer and the different difference of amplification template.Simultaneously, amplification blank.Blank except do not add template in amplification reaction solution, remaining amplification condition all detects the same with the detection of sample.
4. mark pcr amplification product, the part of getting pcr amplification product is in PCR labeled reactant system, add the specific PCR primer and add marker, wherein the sample of Jian Ceing is used different marker marks respectively with blank, such as sample Cy5 mark, and blank Cy3 mark.The mark cycle number is 11 mixing.
5. hybridization, the amplification label product of test sample and blank amplification label product are respectively got equivalent (2-5ul) and are hybridized, and hybridization conditions is consistent with ordinary method.
6. signal detection, it is consistent with ordinary method to hybridize later elution requirement.Slice, thin piece behind the wash-out detects with laser scanner scans.
7. hybridization signal analysis, calculate the signal ratio in each site according to following formula:
ration＝ΔF1/ΔF2
Δ F1 is that the difference of sample marker signal subtracting background signal is (during such as the Cy5 mark in the formula, be F635-B635), Δ F2 is that the difference of the marking signal and the background signal of blank is (such as with the Cy3 mark time, be F532-B532). with the template DNA of equivalent, with identical amplification label condition, and with after carrying out above-mentioned detection behind the different marks, calculate the Ration mean value of two kinds of marking signals, the Cut off value of this value for judging.During sample detection, the ration value in chip site is positive greater than Cut off value, and it is negative to be lower than Cut off value.
Claims (1)
1. clinical infectious disease gene chip diagnosis scheme, this scheme is characterised in that:
1.1 clinical sample DNA is carried out the multiplex PCR amplification with the specific PCR primer, carries out the multiplex PCR amplification label with the specific PCR primer then;
1.2. in sample detection, make a pcr amplification blank, and use the marker mark different with the sample detection mark, hybridize detection with chip then with after the sample marker balanced mix, the ratio of the hybridization signal of the hybridization signal of marker and blank determines whether sample has positive signal per sample;
1.3. determine the pcr amplification cycle number, make the intensity of detection signal become positive correlation, thereby can carry out sxemiquantitative with the copy number of template.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA02128069XA CN1508264A (en) | 2002-12-18 | 2002-12-18 | Clinical infectious disease gene chip diagnosis scheme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA02128069XA CN1508264A (en) | 2002-12-18 | 2002-12-18 | Clinical infectious disease gene chip diagnosis scheme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1508264A true CN1508264A (en) | 2004-06-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CNA02128069XA Pending CN1508264A (en) | 2002-12-18 | 2002-12-18 | Clinical infectious disease gene chip diagnosis scheme |
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| CN (1) | CN1508264A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101541979B (en) * | 2007-06-01 | 2014-11-05 | 科学与工业研究委员会 | A novel method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system |
-
2002
- 2002-12-18 CN CNA02128069XA patent/CN1508264A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101541979B (en) * | 2007-06-01 | 2014-11-05 | 科学与工业研究委员会 | A novel method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system |
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