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Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection primer group, detection probe group, detection kit and detection method for detecting hepatitis B virus (HBV) pregenome ribonucleic acid (pgRNA)

A technology for real-time fluorescent quantitative and detection probes, applied in the field of probe sets, real-time fluorescent quantitative RT-PCR detection primer sets, and kits, can solve problems that cannot meet actual needs, improve detection accuracy, and restore calibration activity , to avoid the effect of low specificity

Active Publication Date: 2017-07-04
GUANGZHOU SUPBIO BIO TECH & SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The sensitivity of the existing technology for the detection of HBV pgRNA still cannot meet the actual needs, and its sensitivity is only 1×10 3 copies / ml around

Method used

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  • Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection primer group, detection probe group, detection kit and detection method for detecting hepatitis B virus (HBV) pregenome ribonucleic acid (pgRNA)
  • Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection primer group, detection probe group, detection kit and detection method for detecting hepatitis B virus (HBV) pregenome ribonucleic acid (pgRNA)
  • Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection primer group, detection probe group, detection kit and detection method for detecting hepatitis B virus (HBV) pregenome ribonucleic acid (pgRNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1 A kind of detection primer set and detection probe set for detecting HBV pgRNA

[0072] The detection primer set includes the following 4 primers:

[0073] Primer F1: its nucleotide sequence is 5'-TGG TGT CTT TTG GAG TGT GGA T-3' (SEQ ID NO: 1),

[0074] Primer R1: its nucleotide sequence is 5'-CCA CCT TAT GTG TCC AAG GAA TAC T-3' (SEQ ID NO: 2),

[0075] Primer F2: its nucleotide sequence is 5'-TTC ACC TCA CCA TAC GGC ACT CAG GC-3' (SEQ ID NO: 3),

[0076] And primer R2: its nucleotide sequence is 5'-ATG AAT GTC AGG AAA AGA AGG AGT TTG CC-3' (SEQ ID NO: 4).

[0077] The detection probe set includes the following two probes:

[0078] Probe P1: its nucleotide sequence is 5'-CGC ACT CCT CCT GCA TAT AGA CCA TCA AA-3' (SEQ ID NO: 5), its 5' end is marked with a fluorescent group FAM, and its 3' end is marked with The quenching group BHQ1;

[0079] And probe P2: 5'-TCT CAA TCG CCG CGT CGC A-3' (SEQ ID NO: 6), its 5' end is marked with a fluorescent group FA...

Embodiment 2

[0081] Embodiment 2 A kind of detection primer set and detection probe set for detecting HBV pgRNA

[0082] The detection primer set includes the following 4 primers:

[0083] Primer F3: its nucleotide sequence is 5'-TTT GGT GTC TTT TGG AGT GTG GA-3' (SEQ ID NO: 7),

[0084] Primer R3: its nucleotide sequence is 5'-CTT ATG TGT CCA AGG AAT ACT AA-3' (SEQ ID NO: 8),

[0085] Primer F4: its nucleotide sequence is 5'-GTT CAC CTC ACC ATA CGG CAC TCA GG-3' (SEQ ID NO: 9),

[0086] And primer R4: its nucleotide sequence is 5'-TGA ATG TCA GGA AAA GAA GGA GTT TGC CA-3' (SEQ ID NO: 10).

[0087] The detection probe set includes the following two probes:

[0088] Probe P3: its nucleotide sequence is 5'-TTC GCA CTC CTC CTG CAT ATA GAC CAT CA-3' (SEQ ID NO: 11), its 5' end is marked with the fluorescent group FAM, and its 3' end is marked with The quenching group BHQ1;

[0089] And probe P4: its nucleotide sequence is 5'-CTC AAT CGC CGC GTC GCA-3' (SEQ ID NO: 12), its 5' end is marked...

Embodiment 3

[0091] Embodiment 3 A kind of kit for detecting HBV pgRNA

[0092] The kit includes:

[0093] (1) detection primer set and detection probe set in embodiment 1;

[0094](2) Enzyme system: 1) Tfl DNA polymerase; 2) MMLV reverse transcriptase; 3) Stoffel fragment;

[0095] (3) Reagent: 1) Tris-sulfuric acid; 2) MOPS buffer; 3) sodium citrate; 4) (NH 4 ) 2 SO 4 ;5) MgSO 4 ; 6) polyoxyethylene lauryl ether (Brij-35); 7) acetylated BSA;

[0096] (4) Negative control substance: physiological saline.

[0097] (5) Positive control substance: the amplified fragment obtained by amplifying the HBV genome with the detection primer set in Example 1, the length of the amplified fragment is 512bp.

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Abstract

The invention belongs to the technical field of biology, and relates to the field of virus detection, in particular to a real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection primer group, a detection probe group, a detection kit and a detection method for detecting hepatitis B virus (HBV) pregenome ribonucleic acid (pgRNA). The detection primer group and the detection probe group comprise a primer group 1 and a probe group 1, or a primer group 2 and a probe group 2. An amplification method, provided by the invention, of double polymerases, i.e., Stoffel fragment and Tf1 deoxyribonucleic acid (DNA) polymerase can realize the amplification for samples which are difficult to amplify normally by the conventional detection method, so that the method has higher sensitivity and accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically relates to the field of virus detection, and more specifically relates to a primer set, probe set, kit and method for real-time fluorescent quantitative RT-PCR detection of HBV pgRNA. Background technique [0002] Hepatitis B virus (HBV) infection is prevalent worldwide, with more than two billion infected people. It is one of the most serious infectious diseases in my country. The 2006 national epidemiological survey of hepatitis B showed that there are still about 93 million hepatitis B surface antigen carriers in my country, of which about 20 million are chronic hepatitis B patients, and hepatitis B and liver cancer patients account for about a quarter of the world. Chronic HBV infection is the main cause of chronic hepatitis, liver cirrhosis, liver failure and primary liver cancer, and causes more than 1 million deaths every year, causing great social harm. [0003] Studies have sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/706C12Q2531/113C12Q2563/107
Inventor 张晓玮彭春梅张嘉邓可基李海茵谢丽娟李家导乐小炎林志豪罗园香张新陈观芝陈凤英林敏深石壮壮林若琳王星王法余培煜莫静嫣
Owner GUANGZHOU SUPBIO BIO TECH & SCI
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