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Kit and method for real-time fluorescence quantitative detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in serum

A real-time fluorescence quantification and detection kit technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of inability to accurately quantify HBV DNA, the operation is not simple enough, and the manufacturing cost is low, so as to reduce medical waste. production, reduce pollution, and improve accuracy

Active Publication Date: 2018-11-13
GUANGZHOU SUPBIO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNA lysate used in this invention is a conventional lysate, the operation is relatively not simple enough, and the time-consuming is relatively long
[0006] There is also a one-step HBV fluorescent quantitative method such as Hunan Shengxiang Biotechnology on the market. For example, Chinese patent 200910309980.2 discloses a one-step fluorescent quantitative PCR detection method for pathogenic nucleic acid. A conservative gene sequence is selected for the nucleic acid of the pathogen to be detected, and specific primers are designed to detect it. Needle, using a nucleic acid rapid release reagent to extract pathogen nucleic acid, directly add the corresponding PCR reaction solution to realize PCR fluorescence quantitative detection, the invention is simple and fast in operation, has strong anti-pollution and anti-interference ability, no loss of extracted nucleic acid, and the manufacturing cost of reagents used Low, but unable to accurately quantify HBV DNA down to 20 IU / mL in serum

Method used

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  • Kit and method for real-time fluorescence quantitative detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in serum
  • Kit and method for real-time fluorescence quantitative detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in serum
  • Kit and method for real-time fluorescence quantitative detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1 A kind of real-time fluorescent quantitative detection kit A of HBV DNA in serum

[0081] The kit includes:

[0082] Detection primer set for hepatitis B virus HBV;

[0083] Detection probe for hepatitis B virus HBV;

[0084] and lysate;

[0085] The lysate comprises 5% (V / V) dodine, 6% (V / V) Tween-20, 20mM sodium citrate and 20mMNaCl;

[0086] The detection primer set of described hepatitis B virus HBV is made up of following primer sequence:

[0087] Primer sequence 1: GGCAACGGCCTGGTCTGTGCCAAGTGT,

[0088] Primer sequence 2: GGCAACGGTCAGGTCTCTGCCAAGTGT,

[0089] Primer sequence 3: TGACGCAACCCCCACTG,

[0090] Primer sequence 4: GCTGCGAGCAAAACAAG,

[0091] Primer sequence 5: GCGCAGGATCCAGTTGGCAGCACA,

[0092] Primer sequence 6: GTCCCGCGCAGGATCCAGTTGGCAGC;

[0093] The detection probe sequence of the hepatitis B virus HBV is: CCGATCCATACTGCGGAACT;

[0094] The fluorescent group labeled at the 5' end of the detection probe sequence of the hepatitis ...

Embodiment 2

[0105] Embodiment 2 A kind of real-time fluorescent quantitative detection kit B of HBV DNA in serum

[0106] Compared with the real-time fluorescent quantitative detection kit A in Example 1, the only difference of the real-time fluorescent quantitative detection kit B in this example is that 25% (V / V) Dodine is used in the lysate.

Embodiment 3

[0107] Embodiment 3 A kind of real-time fluorescent quantitative detection method of HBV DNA in serum

[0108] This embodiment adopts the real-time fluorescent quantitative detection kit A in the embodiment 1 to carry out real-time fluorescent quantitative detection of HBV DNA in serum;

[0109] The real-time fluorescence quantitative detection method comprises the following steps:

[0110](1) Acquisition of serum HBV DNA samples:

[0111] a. Shake the serum test tube for 10 seconds, mix thoroughly, and immediately transfer 200 μL to a 1.5 mL sterilized centrifuge tube, then add 20 μL magnetic beads, 20 μL carrier RNA, and 300 μL lysate, mix by inversion, and incubate at 50°C with shaking 6 minutes;

[0112] b. Transfer the centrifuge tube to the magnetic stand for 6 minutes to collect the magnetic beads, and discard the supernatant;

[0113] c. Briefly centrifuge to collect the liquid droplets on the tube wall, transfer the centrifuge tube to the magnetic stand for 1 minut...

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Abstract

The invention discloses a kit and a method for real-time fluorescence quantitative detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA) in serum. By using dodine to replace the traditionalguanidine salt and part of a decontamination agent in the lysate, the lysate can effectively lyse a serum sample containing HBV particles so as to release the HBV DNA in the serum sample; therefore,the kit and the method remarkably increases the lysis efficiency and greatly shortens the detection time.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time fluorescent quantitative detection kit for HBV DNA in serum and a detection method thereof Background technique [0002] Hepatitis B virus, referred to as hepatitis B virus, is a DNA virus. According to current knowledge, HBV is only susceptible to humans and chimpanzees, causing hepatitis B viral hepatitis disease. The whole hepatitis B virus is granulated, also known as Dana granules (Dane). Discovered by Dana in 1965, the diameter is 42 nanometers, and the particles are divided into two parts: the shell and the core. The genome of hepatitis B virus (HBV) is about 3.2kb in length and is partially double-stranded circular DNA. The infection rate of hepatitis B virus in China is about 60%-70%, and the carrier rate of hepatitis B surface antigen accounts for about 7.18% of the total population. Based on this calculation, there are about 93 milli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/6806
CPCC12Q1/6806C12Q1/6851C12Q1/706C12Q2527/125C12Q2563/143C12Q2563/149C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 张晓玮林若琳罗园香杨芬石壮壮彭春梅张嘉邓可基李家导陈观芝蔡佺佑蔡培宏颜道宇王旭川黄彩兰李思成邢兰坤王星王法
Owner GUANGZHOU SUPBIO BIO TECH & SCI
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