Kit for detecting proviral DNA (Deoxyribonucleic Acid) of HIV (Human Immunodeficiency Virus)

A kit and provirus technology, applied in the field of ring-mediated constant temperature amplification detection kits, can solve problems such as applicability limitations

Inactive Publication Date: 2013-09-04
杭州市儿童医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these technical professional instruments an

Method used

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  • Kit for detecting proviral DNA (Deoxyribonucleic Acid) of HIV (Human Immunodeficiency Virus)

Examples

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Embodiment 1

[0014] Example 1: Loop-mediated constant temperature amplification combined with fluorescent dye technology to detect HIV proviral DNA specificity and sensitivity experiment

[0015] 1. Materials:

[0016] Select 10 cases of positive samples of HBV, HCV, UU, CT, HSV, etc. that cause clinical viral infections, and construct HIV amplification-positive plasmids at the same time, and conduct sensitivity experiments after sequentially 10-fold dilution of the specific plasmids. All strains were provided by the Bacteria Laboratory of the Sixth People's Hospital of Hangzhou.

[0017] 2. Loop-mediated constant temperature amplification combined with fluorescent dye technology to detect HIV proviral DNA specificity:

[0018] The specificity of this set of primers was evaluated, and 10 cases of HBV-DNA, HCV-RNA, Uu-DNA, CT-DNA, and HSV-DNA positive samples were used for HIV-1 DNA detection, and the results were all negative, that is, this method 100% specificity;

[0019] 3. Loop-...

Embodiment 2

[0021] Example 2: Clinical application of loop-mediated constant temperature amplification combined with fluorescent dye technology for detection of HIV proviral DNA

[0022] 1. Specimen testing:

[0023] The selected 200 HIV-infected patients were all inpatients and outpatients of Hangzhou Sixth People's Hospital from April 2011 to December 2012. All patients were diagnosed with HIV by Hangzhou (or Zhejiang Province) Center for Disease Control and Prevention infected. The age distribution ranged from 19 to 62 years old, with an average age of 34.5 years. Among them, there were 145 males and 55 females. 50 cases of healthy control group were the blood samples of the employees of our hospital.

[0024] 2. Control detection kit:

[0025] The HIV-1 RNA detection kit is carried out using the real-time fluorescent RT-PCR detection HIV-RNA kit produced by Sun Yat-Sen University Daan Gene Co., Ltd., which has been certified by the national SFDA. According to the RNA extraction me...

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Abstract

The invention discloses a kit for detecting the proviral DNA (Deoxyribonucleic Acid) of an HIV (Human Immunodeficiency Virus). An isothermal amplification reagent is placed in the kit and contains an aqueous solution of primers LTR-F3, LTR-B3, LTR-FIP and LTR-BIP, an aqueous solution of MgCl2, an aqueous solution of dNTP, a 10*betaine buffer, a 20*SYBR dye, a BstDNA polymerase and 3.9 microliters of sterile water for injection. According to the kit, after a loop-mediated isothermal amplification reaction system is successfully synthesized, the system is combined with a fluorescent dye, the SYBR Green I fluorescent dye is combined with amplified fragments along with the amplification, the fluorescence intensity is recorded by a fluorescent quantitative PCR (Polymerase Chain Reaction) instrument, and finally, the virus content is given according to the fluorescence intensity.

Description

technical field [0001] The invention relates to a detection kit for loop-mediated constant temperature amplification, in particular to a method for quantitatively detecting the HIV proviral gene (HIV) after integration of HIV virus and host cell chromosomal DNA by combining loop-mediated constant temperature amplification technology with fluorescent dye technology Kit for Proviral DNA. Background technique [0002] With the development of molecular biology technology, the application of polymerase chain reaction (PCR), in situ hybridization and other technologies has greatly improved the sensitivity and specificity of HIV detection. However, these technical professional instruments and operators have certain limitations in applicability. Japanese scholar Notomi et al. invented a new nucleic acid amplification technology-loop-mediated constant temperature amplification technology. The reaction relies on 4 specific primers that can recognize 6 specific regions on the target D...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 厉小玉吴亦栋韦翊
Owner 杭州市儿童医院
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