Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid

A hepatitis virus, quantitative detection technology, applied in the field of biomedical clinical diagnosis, can solve the problems of complex operation, poor accuracy, low sensitivity, etc., to achieve high sensitivity, avoid nucleic acid loss, and optimize the effect of PCR reaction system

Active Publication Date: 2012-12-19
东北制药集团辽宁生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current quantitative kit technologies still have disadvantages such as complicated operation, low sensitivity, and poor accuracy, and the detection technology needs to be improved and improved.

Method used

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  • Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid
  • Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid
  • Kit and detection method for rapid quantitative detection of hepatitis virus nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The detection of embodiment 1 HBV negative and positive serum

[0047] Take 5 μl of nucleic acid release agent (0.25% SDS, 0.25% Chaps, 5% (NH 4 ) 2 SO 4 , composed of 1% formamide. ) into the PCR reaction tube. Then draw 5 μl of serum samples to be tested, positive control, negative control, HBV DNA positive virus serum (1000IU / ml, 500IU / ml) of known concentration, join in the nucleic acid releasing agent, and pipette gently sucks for 5 -8 times, rest at room temperature for 10 minutes. Then add 1 μl internal standard (known positive internal control-500copies / μl) to each tube according to the number of reactions, and finally add 40 μl of the prepared PCR reaction solution. The composition of the reaction solution is as follows, and perform fluorescence quantitative PCR on Bio-Rad IQ5 Amplification, the procedure is as follows.

[0048] PCR reaction solution components

[0049] Element

Dosage

10×Buffer

5.0μl

dNTPS

5.0μl

...

Embodiment 2

[0058] Embodiment 2. comparative analysis of the present invention and the kits of mainstream manufacturers

[0059] Use this kit and kits from domestic mainstream manufacturers to detect 3 HBV DNA positive sera at the same time. The operating method of the present invention is the same as above. The specific process of kits from mainstream manufacturers is as follows: take 100 μl of the sample to be tested, the positive control, the negative control, and 3 parts of HBV DNA positive serum with a filter tip, and add them to the centrifuge tube, then add 100 μl of sample treatment solution A, shake and mix. uniform. Centrifuge at 12000 rpm for 10 minutes, then discard the supernatant, add 25 μl of sample treatment solution B to the pellet, and pipette 8-10 times. Boil the centrifuge tube in boiling water at 100°C for 10 minutes, then take out the centrifuge tube and centrifuge at 12,000 rpm for 10 minutes, and finally take 2 microliters of the supernatant for PCR amplification...

Embodiment 3

[0063] Example 3 Rapid detection of HCV clinical samples

[0064] Take 5 μl of nucleic acid release agent (0.25% SDS, 0.25% Chaps, 5% (NH4) 2 SO 4 , composed of 1% formamide. ) into the PCR reaction tube. Then take 5 μl of the serum samples to be tested, positive control, negative control and 4 copies of HCV RNA positive virus serum of known concentration, add them to the nucleic acid release agent, pipette gently for 5-8 times, and stand at room temperature for 10 minute. Then add 1 μl internal standard (known positive internal control-500copies / μl) to each tube according to the number of reactions, and finally add 40 μl of the prepared PCR reaction solution. The composition of the reaction solution is as follows, and perform fluorescence quantitative PCR on Bio-Rad IQ5 Amplification, the amplification procedure is as follows.

[0065] PCR reaction solution components

[0066] Element

Dosage

10×Buffer

5.0μl

dNTPS

5.0μl

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Abstract

The invention relates to the field of biomedical clinical diagnosis, and specifically relates to a kit and a detection method for rapid quantitative detections of hepatitis virus nucleic acids. The kit comprises a nucleic acid releasing reagent, a nucleic acid amplification reagent, a control substance, a standard substance, an internal standard, and a template. The nucleic acid releasing reagent is a water solution of SDS, 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate (Chaps), (NH4)2SO4, and formamide. According to volume percentages, the nucleic acid releasing reagent comprises 0.25% of SDS, 0.25% of Chaps, 5% of (NH4)2SO4, 1% of formamide, and balance of water. The invention has the advantage that the invention provides a releasing reagent which can highly efficiently and rapidly release hepatitis virus nucleic acid, and an application thereof. Under the effect of the nucleic acid releasing reagent, the nucleic acid can be directly released from trace samples, and can be directly added into a PCR reaction liquid for fluorescent quantitative detection. Therefore, rapid nucleic acid detection is realized. Sample nucleic acid is not required to be extracted independently. Nucleic acid extraction and fluorescence quantitative PCR detection are directly achieved in a PCR reaction tube.

Description

technical field [0001] The invention relates to the field of biomedical clinical diagnosis, in particular to a kit and a detection method for rapid and quantitative detection of hepatitis virus nucleic acid. Background technique [0002] The pathogenic microorganisms of many epidemic infectious diseases in our country, such as AIDS, viral hepatitis, tuberculosis, syphilis, etc., have caused great harm to human health. Clinically, methods such as immunology and microbiology are mainly used to detect and diagnose various pathogenic microorganisms. Due to the limitations of low sensitivity and long cycle, it is still impossible to completely eliminate the missed detection of positive pathogens and timely and accurate diagnosis. In recent years, nucleic acid molecular detection technology has shown strong advantages in laboratory medicine and clinical research. Compared with immunodiagnostic technology, it has the advantages of good sensitivity, strong specificity and quantifica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 李望丰李静芝周凯许守民刘立侠
Owner 东北制药集团辽宁生物医药有限公司
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