Reagent kit for synchronously detecting hepatitis, AIDS virus and syphilis helicoid nucleic acid

A technology for Treponema pallidum and HIV, applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve problems such as lack of promotion and popularization, impact on blood safety, lack of simultaneous quantitative detection technology and products for four kinds of pathogenic nucleic acids , to achieve the effect of excellent specificity and excellent sensitivity

Active Publication Date: 2013-11-27
WUHAN ZHENFU PHARMA CO LTD
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

However, although the NAT method has obvious advantages in ensuring blood safety, so far, this technology has not been promoted and popularized in China
Moreover, the foreign situation is not completely applicable to the domestic situation. The foreign NAT method mainly targets the three viruses of HIV, HBV, and HCV. These three viruses are the biggest scourge affecting the safety of blood abroad; Syphilis detection must be carried out, because with the reform and opening up and changes in domestic concepts, the incidence of syphilis is on the rise, and it is also the chief culprit affecting blood safety.
At present, although foreign countries have developed and developed NAT methods and reagents for the three viruses, and some companies are applying for approval of related products, there is no technology and product for the simultaneous quantitative detection of these four pathogenic nucleic acids at home and abroad.

Method used

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  • Reagent kit for synchronously detecting hepatitis, AIDS virus and syphilis helicoid nucleic acid
  • Reagent kit for synchronously detecting hepatitis, AIDS virus and syphilis helicoid nucleic acid

Examples

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Embodiment 1

[0059] One, embodiment 1: kit composition and preparation

[0060] 1. Synchronous extraction of pathogen concentrate and DNA and RNA

[0061] For self-preparation, pathogen concentrate: 20% PEG6000 volume fraction in normal saline solution. DNA and RNA synchronous extraction solution: the final concentration is 4mol / L guanidine isothiocyanate, the volume fraction is 0.5% sodium lauryl sarcosinate, 0.1mmol / L β-mercaptoethanol, 25mmol / L sodium citrate, 0.20g / L glycogen.

[0062] 2. Fluorescent PCR reaction solution

[0063] .20.0mmol / L MgSO 4 The 10×buffer, the forward and reverse primers of hepatitis B virus, hepatitis C virus, HIV and Treponema pallidum are 10 μmol / L respectively, and the fluorescent probes of hepatitis B virus, hepatitis C virus, HIV and Treponema pallidum are respectively is 10μmol / L, 25mmol / L

[0064] MgCl 2 , 10mmol / L dNTPs mix and sterile double distilled water.

[0065] 3. Standard positive template storage solution

[0066] Concentration is 10 ...

Embodiment 2

[0068] 2. Example 2: Synchronous detection of hepatitis B, hepatitis C, HIV and Treponema pallidum using kits

[0069] 1. Put 100 μl of body fluid specimen into a 1.5ml centrifuge tube, add an equal volume of pathogen concentrate, shake well, centrifuge at 10,000 g for 15 minutes, add 50 μl of DNA and RNA synchronous extraction solution to the precipitate, mix well, and place at 37°C or room temperature for 15 minutes; Then add 100ul-20°C pre-cooled isopropanol, precipitate at 4°C for 15min, centrifuge at 10,000g for 15min, discard the supernatant; add 500ul of 75% ethanol to the precipitate, invert several times to wash the remaining isopropanol, centrifuge at 13,000rpm for 15min, Gently pour off the supernatant; centrifuge at 3000rpm for 10sec, shake off the remaining liquid on the tube wall to the bottom, blot dry with a micropipette, and dry at room temperature for 2-3min (do not overdry to prevent insoluble nucleic acid in the next step); add 40ul DEPC water ( Add DEPC pu...

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Abstract

The invention discloses a kit which can synchronously diagnose hepatitis, AIDS virus and treponema pallidum nucleic acid, and relates to a gene diagnosis kit. The invention comprises: (1) pathogene concentrated solution and DNA and RNA nucleic acid simultaneous extraction liquid; (2) positive plasmid with the diagnose sequence of four pathogens comprising hepatitis b virus, hepatitis c virus, AIDS virus and treponema pallidum; and (3) TaqMan PCR amplified protocol diagnosed by the combination of one-step monosiphonous and single enzyme. The invention carries out simultaneous and real-time diagnosis, and has the advantages of having short flow time, high efficiency and high sensitivity, and avoiding the possible pollution and artificial errors owing to the simple operation and close diagnosis,. The kit is suitable to large-scale and high-flux blood screening, such as the nucleonic acid screening in blood center and biological product manufacturer, and is also suitable to high-capacity and high-efficient clinical nucleic acid examination.

Description

technical field [0001] The invention relates to a genetic diagnosis kit, in particular to a highly sensitive, specific and suitable for synchronous extraction and synchronous real-time detection of hepatitis (including hepatitis B and hepatitis C), HIV And a test kit for Treponema pallidum nucleic acid. Background technique [0002] AIDS, hepatitis C, hepatitis B and syphilis are all major infectious diseases transmitted through blood, and blood transfusion has always been one of the main routes of transmission of the above diseases. The first clinically reported AIDS patient in my country was infected with HIV through transfusion of unhealthy blood products. The latest statistics from the World Health Organization show that nearly one million people in the world are infected with infectious diseases such as AIDS and viral hepatitis every year due to the transfusion of unhealthy blood or blood products; 5% to 10% of the newly added HIV carriers each year are transfused or ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
Inventor 王业富周立唐景峰龚路路
Owner WUHAN ZHENFU PHARMA CO LTD
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