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HIV gene detecting chip and its prepn process and usage detecting HIV nucleic acid

A gene detection chip and gene fragment technology, applied in the field of HIV nucleic acid detection, can solve the problems of cumbersome operation, increased cost and cross-contamination, difficult to popularize and apply basic clinical detection, etc. Opportunities, effects of improving quality and usability

Inactive Publication Date: 2007-10-03
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only cumbersome to operate, but also increases cost and chance of cross-contamination
Therefore, this method is difficult to be promoted and applied in primary clinical testing

Method used

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  • HIV gene detecting chip and its prepn process and usage detecting HIV nucleic acid
  • HIV gene detecting chip and its prepn process and usage detecting HIV nucleic acid

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Effect test

preparation example Construction

[0025] The preparation method of the above-mentioned HIV gene detection chip provided by the embodiment of the present invention, its steps include:

[0026] (1) Add a reaction system containing random hexanucleotide primers in a test tube, the reaction system also contains primer sets SEQ ID NO.14+12, SEQ ID NO.20+10, SEQ ID NO.20+11 , SEQ ID NO.21+13, SEQ ID NO.18+12, SEQ ID NO.15+12, SEQ ID NO.20+9, SEQ ID NO.16+9, SEQ ID NO.17+10, SEQ ID NO.17+10, SEQ ID NO. One or more of ID NO.19+11, complete reverse transcription reaction and PCR amplification for HIV RNA samples at one time;

[0027] (2) Pick positive fragment clones from the fragments obtained above, and save them after sequencing confirmation;

[0028] (3) After the positive fragment primer combination of the above clone is confirmed by RT-PCR in the blood of different patients, select a primer combination probe with high specificity and high sensitivity, that is, a primer with the sequence of SEQ ID NO: 1-6 Combin...

Embodiment 1

[0040] Embodiment 1: Preparation of HIV gene detection chip

[0041] Obtain HIV-specific nucleic acid fragments from HIV-infected persons, clone and store them after verification, and use them as probes for HIV gene detection chips. This method can be subdivided into:

[0042] (1) Add a reaction system containing random hexanucleotide primers in a test tube, the reaction system also contains primer sets SEQ ID NO.14+12, SEQ ID NO.20+10, SEQ ID NO.20+11, SEQ ID NO.21+13, SEQ ID NO.18+12, SEQ ID NO.15+12, SEQ ID NO.20+9, SEQ ID NO.16+9, SEQ ID NO.17+10, SEQ ID NO. One or more of 19+11, complete reverse transcription reaction and PCR amplification for HIV RNA samples at one time;

[0043] Among them, the above-mentioned SEQ ID NO: 14-21 sequence is an oligonucleotide forward primer for HIV-specific fragments; the above-mentioned SEQ ID NO: 9-13 sequence is an oligonucleotide reverse primer for HIV-specific fragments; random six Nucleotide primers refer to primers with a length ...

Embodiment 2

[0097] Embodiment 2: HIV nucleic acid detection method

[0098] (A) HIV RNA is isolated from the sample by:

[0099] Prepare:

[0100] Dissolve Carrier RNA with 1ml SKVR (Solution I) binding solution, and transfer to Solution I bottle.

[0101] Divide the Solution I solution dissolved in Carrier RNA into small portions, and store in a refrigerator at 4°C for 6 months. If there is precipitation, heat at 37°C before use, and do not heat the same reagent more than 5 times.

[0102] Add an equal volume of absolute ethanol to SKVR wash solution A.

[0103] Add absolute ethanol to SKVR washing solution B to a final ethanol concentration of 35%.

[0104] operate:

[0105] Take a 1.5ml centrifuge tube, add 300μl SKVR binding solution, then add 200μl plasma, gently suck 4 times with a micropipette, and let stand at room temperature for 10 minutes.

[0106] Take 200 μl of DEPC-treated water and put it in a small centrifuge tube, put it in a 65°C water bath, and preheat it for late...

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Abstract

The present invention relates to HIV nucleic acid detection, and is especially one kind of HIV gene detecting chip and its preparation process and usage in detecting HIV nucleic acid. The chip has solid carrier, and specific HIV gene segment including SEQ ID Nos. 1-6 sequences, positive contrast of SEQ ID No. 7 sequence, and negative contrast of SEQ ID No. 8 sequence fixed on the carrier. The chip is used for detecting HIV nucleic acid, and has high sensitivity, high specificity, high product quality and high practicability.

Description

technical field [0001] The invention relates to a human immunodeficiency virus (HIV) gene detection chip, its preparation method and its application method for detecting HIV nucleic acid. Background technique [0002] Acquired Immunodeficiency Syndrome (AIDS) is a systemic disease caused by Human Immunodeficiency Virus (HIV). In just 20 years, it has caused very serious harm to the whole world, affecting almost all countries and regions in the world. Laboratory diagnosis of HIV includes etiological diagnosis and immunological detection, and the most commonly used detection method (including primary screening and diagnosis) is antibody detection. This method has the advantages of strong specificity and high sensitivity, but since the detection is the specific antibody against HIV produced in the human body, it is necessary to wait until the HIV virus reproduces in the body for a period of time to stimulate the body to produce a certain titer of antibody, to be detected. In ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 冯铁建赵广录王晓辉陈琳石向东
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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