New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.029

Abstract

Abstract: Our purpose is to eliminate the false positive problem caused by primer-probe aggregation and extension,as well as the false negative problem caused by primer dimer（PD）. First,the primer and probe aggregation results were tested for different probes;then the appropriate internal control gene（IC）was selected based on the competitive interference experiments of duplex PCR,simultaneously,the practicability of central-home primer excluding the inference of PD was verified on the plasmid of the IC;and finally,the sensitivities of different systems for TaqMan method were compared. The probe HBVP4,containing an antisense base,did not produce false positive results in the repeated trials;the concentration difference at which interferences for the templates of competitive and non-competitive duplex PCR were noticed was 20 times and 100 times,respectively. The dilution that could detected by central-home primer and common primer was 10^-9 and 10^-8,respectively. And for 3 HBV gene detection systems,the detection could be until Ct33 if using common primer,and Ct35 if using central-home primer and by the accession of IC. Based on adjustments of TaqMan-Molecular Beacon and introducing antisense,the false positive problem caused by primer-probe aggregation and extension was excluded. In probe method,the use of central-home primer and IC could both reduced the influences caused by PD,and therefore increase the sensitivity of detection.

Key words: real time fluorescent quantitative PCR, TaqMan probe, antisense base, primer dimer, internal control

JIANG Wen-can, YUE Su-wen, JIANG Hong, GE Su-jun, WANG Cheng-bin. New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR[J]. Biotechnology Bulletin, 2016, 32(11): 107-114.

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