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.
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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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