1. A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.
- Author
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Han Y, Hou SY, Ji SZ, Cheng J, Zhang MY, He LJ, Ye XZ, Li YM, and Zhang YX
- Subjects
- Base Sequence, DNA Probes chemistry, DNA Probes metabolism, HIV genetics, Hepacivirus genetics, Humans, Phase Transition, RNA, Viral genetics, RNA, Viral metabolism, Reverse Transcriptase Polymerase Chain Reaction, RNA, Viral analysis, Real-Time Polymerase Chain Reaction
- Abstract
A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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