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Real-time fluorescent RT-PCR detection method and kit for Mycobacterium tuberculosis 85b mRNA

A Mycobacterium tuberculosis, RT-PCR technology, applied in the field of molecular biology, can solve the problems of difficult mRNA extraction, long half-life, high detection limit, etc.

Active Publication Date: 2020-09-29
GUANGZHOU SUPBIO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] These DNA-based in vitro amplification detection methods have the same problem: DNA is a stable nucleic acid molecule with a long half-life, DNA-based in vitro amplification, and its template can come from either live bacteria or dead bacteria
Compared with ordinary PCR, real-time fluorescent quantitative PCR has higher sensitivity, but its specificity is relatively general, and it is easy to affect the stability of detection results due to the inhibitory effect of inhibitors in the sample
In addition, because the mRNA is not easy to extract, if the extraction method using Trizol plus glass beads is used, the method published in this patent application can only detect Mycobacterium tuberculosis specimens with a bacterial count of 15,000 per milliliter, and its detection limit is relatively high, which is difficult to adapt to clinical need

Method used

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  • Real-time fluorescent RT-PCR detection method and kit for Mycobacterium tuberculosis 85b mRNA
  • Real-time fluorescent RT-PCR detection method and kit for Mycobacterium tuberculosis 85b mRNA
  • Real-time fluorescent RT-PCR detection method and kit for Mycobacterium tuberculosis 85b mRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 is used to detect the kit of Mycobacterium tuberculosis 85B mRNA

[0071] The kit includes: a detection primer set and a detection probe for Mycobacterium tuberculosis 85B mRNA;

[0072] Among them, the detection primer set includes: primer TB 85B mRNA-F1: 5'-TTC ACA GCG GCA CAA CAA TATGTC-3'; primer TB 85B mRNA-R1: 5'-GAC TGT GCG TTC CTG ATC GAG-3'; primer TB 85B mRNA-F2: 5'-CGG TTT ATT TCG ACA GCG AAG AC-3'; Primer TB 85B mRNA-R2: 5'-AAC TGG TTT CGCACC GTG TC-3'.

[0073] The detection probe set includes: probe TB 85B mRNA-P1: 5'-AAC CAT GCA ATG ATG CTC GT-3'; probe TB 85B mRNA-P2: 5'-CAA TGA TGC TCG TGC AAC ACC T-3' ; The 5' end of the nucleotide sequence of probe TB 85B mRNA-P1 and probe TB 85B mRNA-P2 is connected with fluorescent group FAM, and the 3' end is connected with quenching group BHQ1.

Embodiment 2

[0074] Embodiment 2 is used for detecting the kit of Mycobacterium tuberculosis 85B mRNA

[0075] The kit includes: a detection primer set and a detection probe for Mycobacterium tuberculosis 85B mRNA;

[0076] Among them, the detection primer set includes: primer TB 85B mRNA-F1': 5'-CAC AGC GGC ACA ACA ATATGT CG-3'; primer TB 85B mRNA-R1': 5'-ACT GTG CGT TCC TGA TCG AGC-3' ; Primer TB 85B mRNA-F2': 5'-TTT ACA GCC AAG CGG TCG AG-3'; Primer TB 85B mRNA-R2': 5'-CGC CGG GAATTT CGA CAC GA -3'.

[0077] The detection probe set includes: probe TB 85B mRNA-P1': 5'ATG ATG CTC GTG CAA CAC CTG C-3'; probe TB 85B mRNA-P2': 5'-ACG AAA CCA TGC AAT GAT GCT CGT GC -3'; the 5' end of the probe TB 85B mRNA-P1' and the probe TB 85B mRNA-P2' nucleotide sequence is connected with a fluorescent group FAM, and the 3' end is connected with a quenching group BHQ1.

Embodiment 3

[0078] Embodiment 3 is used for detecting the kit of Mycobacterium tuberculosis 85B mRNA

[0079] The kit includes:

[0080] (1) detection primer set and detection probe in embodiment 1;

[0081] (2) Enzyme system: Tfl DNA polymerase, MMLV DNA polymerase and Stoffel fragment;

[0082] (3) PCR reagent: Tris-H at pH 8.5 2 SO 4 , 3-(N-morpholino)propanesulfonic acid, pH 7.9, sodium citrate, (NH4) 2 SO 4 , MgSO 4 , Brij-35, acetylated bovine serum albumin and dNTP;

[0083] (4) Positive quality control product: using the genomic DNA of Mycobacterium tuberculosis TB as a PCR template, the 85B mRNA genomic nucleic acid fragment of Mycobacterium tuberculosis TB amplified by detection primer set 1 or detection primer set 2, according to the routine gene MS2 pseudovirus synthesized by engineering method;

[0084] (5) Negative quality control: DEPC treated H 2 O.

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Abstract

The invention belongs to the technical field of molecular biology, particularly relates to the field of detection of mycobacterium tuberculosis, and more particularly relates to a detection primer set, a probe set, a kit and a detection method for a real-time fluorescence RT-PCR (Reverse Transcription-Polymerase Chain Reaction) of mycobacterium tuberculosis 85B mRNA. The detection primer set provided by the invention comprises nucleotide sequences as shown in SEQ ID NO:1 to 4 or nucleotide sequences as shown in SEQ ID NO:7 to 10; the detection probe set comprises nucleotide sequences as shown in SEQ ID NO: 5 and 6 or nucleotide sequences as shown in SEQ ID NO:11 and 12. According to the invention, due to introduction of specific amplification primers and a fluorescence probe as well as an efficient PCR thermal circulation reaction system matched with the specific amplification primers and the fluorescence probe, sensitivity and specificity of detection are reinforced to a great degree. Meanwhile, according to the invention, Tris-MOPS-sodium citrate buffer solution more suitable for RNA (Ribonucleic Acid) amplification and an amplification method adopting double polymerases of a Stoffel segment and a Tfl DNA polymerase are introduced, so that when detection specificity is improved, more templates can be tolerated, and detection sensitivity and stability of a detection result are improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a real-time fluorescent RT-PCR detection method and kit for Mycobacterium tuberculosis 85B mRNA. Background technique [0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). In 2015, it was estimated that there were 10.4 million new cases worldwide, and about 1.5 million people died of tuberculosis. In 2014, the number of new tuberculosis cases in my country was 930,000, ranking third in the world. Tuberculosis has become one of the leading causes of death from infection worldwide. However, there is currently no breakthrough in the research on TB vaccines. Therefore, early diagnosis and early treatment of tuberculosis and blocking the infection of Mycobacterium tuberculosis are particularly important. [0003] To achieve effective prevention and control of tuberculosis, this requires us to have an effective diagnosti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2545/113C12Q2563/107
Inventor 彭春梅林志豪张晓玮邓可基乐小炎张嘉李家导陈观芝林敏深林若琳石壮壮罗园香莫静嫣李海茵张新王星王法吴彩虹
Owner GUANGZHOU SUPBIO BIO TECH & SCI
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