High-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof

A high-sensitivity, quantitative detection technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of no unified detection standard, complex data processing, low sensitivity, etc., and achieve rapid and high detection Sensitivity, easy-to-operate effects

Inactive Publication Date: 2019-12-20
SUZHOU CHIEN SHIUNG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are many methods for quantitative detection of HBV DNA, but there is no unified detection standard. The commonly used quantitative detection method is mainly the fluorescent quantitative PCR method, but the data processing of the fluorescent quantitative PCR method is complicated, requires reference materials or standards, and is easy to detect. Affected by PCR inhibitors, simultaneous photoquantitative PCR method has large error and low sensitivity when measuring low-titer hepatitis B virus

Method used

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  • High-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof
  • High-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof
  • High-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A high-sensitivity HBV DNA digital PCR quantitative detection kit, comprising an upstream primer container, a downstream primer container, a probe container, a digital PCR reaction buffer container, a positive control container and a negative control container;

[0042] Wherein the upstream primer sequence is SEQ ID NO.1=5'-ATGTTGCCCGTTTGTCCTCT-3',

[0043] The downstream downstream primer sequence is SEQ ID NO.3=5'-GCCCTACGAACCACTGAACA-3'

[0044] The probe primer sequence is SEQ ID NO.5=5'-FAM-CCGGACCTGCATGACTACTG-TAMRA-3'.

[0045] The following are the detection steps:

[0046] (1) Dissolve the upstream primer, downstream primer, and probe primer with 1 mM TE buffer at pH 7.4 to obtain a 20 μM solution, and store at 4°C.

[0047] (2) Mix the upstream primer, downstream primer, and probe primer according to a certain ratio. Take 1 μl of probe mixture, 10 μl of 2×ddPCR Supermix without dUTP, 1 μl of sample, and add a certain amount of water to prepare 20 μl of PCR ...

Embodiment 2

[0055] A high-sensitivity HBV DNA digital PCR quantitative detection kit, comprising an upstream primer container, a downstream primer container, a probe container, a digital PCR reaction buffer container, a positive control container and a negative control container;

[0056] Wherein the upstream primer sequence is SEQ ID NO.2=5'-TGGAACCTTTTCGGCTCCTC-3',

[0057] The downstream downstream primer sequence is SEQ ID NO.4=5'-GGGAGTCCGCGTAAAGAGAG-3'

[0058] The probe primer sequence is SEQ ID NO.6=5'-FAM-TCGTTTCCATGGCTGCTAGG-TAMRA-3';

[0059] The following are the detection steps:

[0060] (1) Dissolve the upstream primer, downstream primer, and probe primer with 1 mM TE buffer at pH 7.4 to obtain a 20 μM solution, and store at 4°C.

[0061] (2) Mix the upstream primer, downstream primer, and probe primer according to a certain ratio. Take 1 μl of probe mixture, 10 μl of 2×ddPCR Supermix without dUTP, 1 μl of sample, and add a certain amount of water to prepare 20 μl of PCR ...

Embodiment 3

[0068] A high-sensitivity HBV DNA digital PCR quantitative detection kit, comprising an upstream primer container, a downstream primer container, a probe container, a digital PCR reaction buffer container, a positive control container and a negative control container;

[0069] Wherein the upstream primer sequence is SEQ ID NO.1=5'-ATGTTGCCCGTTTGTCCTCT-3',

[0070] The downstream downstream primer sequence is SEQ ID NO.3=5'-GCCCTACGAACCACTGAACA-3'

[0071] The probe primer sequence is SEQ ID NO.5=5'-FAM-CCGGACCTGCATGACTACTG-TAMRA-3';

[0072] The following are the detection steps:

[0073] (1) Dissolve the upstream primer, downstream primer, and probe primer with 1 mM TE buffer at pH 7.4 to obtain a 20 μM solution, and store at 4°C.

[0074] (2) Mix the upstream primer, downstream primer, and probe primer according to a certain ratio. Take 1 μl of probe mixture, 10 μl of 2×ddPCR Supermix without dUTP, 1 μl of sample, and add a certain amount of water to prepare 20 μl of PCR ...

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Abstract

The invention relates to a high-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof. The high-sensitivity HBV DNA digital PCR quantitative detection kit comprises a forward-primer container, a reverse-primer container, a probe container, a digital PCR buffer solution container, a positive control container and a negative control container. According to the high-sensitivity HBV DNA digital PCR quantitative detection kit, a method of using digital PCR for absolutely quantifying the content of target nucleic acid in samples is utilized for accurately measuring the quantity of HBV DNA, and therefore, the high-sensitivity HBV DNA digital PCR quantitative detection kit can be applied to the fields of biology, agriculture, medicine and the like and has good development prospects especially in the aspect of human hepatitis B virus detection.

Description

technical field [0001] The invention belongs to the field of virus nucleotide detection, and in particular relates to a high-sensitivity HBV DNA digital PCR quantitative detection kit and an application thereof. [0002] technical background [0003] HBV is a double-stranded DNA virus. If hepatitis B virus DNA is detected in the body, the person can be identified as a hepatitis B virus infected person. The amount of HBV DNA is the most reliable method to judge the active degree of HBV replication. Less hepatitis B virus DNA in the blood indicates that the replication of hepatitis B virus is inhibited and the infectivity is weak; more hepatitis B virus DNA in the blood indicates active replication of hepatitis B virus and strong infectivity. Its quantitative detection is of great significance for the formulation of treatment plan and the judgment of treatment effect. [0004] The quantitative nucleic acid detection method developed in recent years can carry out relative qua...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/706C12Q2600/166C12Q2563/159C12Q2563/107C12Q2545/113C12Q2537/16
Inventor 朱志强姚骅珊于苏霞
Owner SUZHOU CHIEN SHIUNG INST OF TECH
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