Multi-temperature single-fluorescence-channel multiple real-time fluorescence quantitative PCR instrument and detection method thereof

Abstract

The invention discloses a multi-temperature single-fluorescence-channel multiple real-time fluorescence quantitative PCR instrument and a detection method thereof. The PCR instrument comprises an optical system, a temperature control system, a fluorescence signal acquisition system and a computer analysis processing system. The temperature control system comprises N annealing temperature control modules; the fluorescence signal acquisition system collects fluorescence intensity corresponding to M primer probe groups at N annealing temperatures at the same time, and the fluorescence signal acquisition system transmits fluorescence signals acquired in real time to the computer analysis processing system; and the computer analysis processing system processes the fluorescence intensity values,and draws a plurality of amplification curves of different primer probes according to the calculated fluorescence intensity values at the N different annealing temperatures. Fluorescent signal collection and data analysis can be carried out at multiple annealing temperatures or extension temperatures, and the design scheme of the primers with different Tm values is matched, so that single-channelmulti-temperature multiple fluorescent quantitative PCR detection is realized.

Description

Technical field[0001]The invention relates to the field of biotechnology detection, in particular to a multi-temperature single-fluorescence channel multiple real-time fluorescence quantitative PCR instrument and a detection method thereof.Background technique[0002]The basic principle of PCR is to use DNA to become single-stranded when it is denatured in vitro. At low temperature, primers and single-strands anneal according to the principle of complementary base pairing, and then adjust the temperature to the optimal reaction temperature for DNA polymerase, and DNA polymerase follows the phosphoric acid The process of synthesizing complementary chains in the direction of carbon-free sugars.[0003]The PCR reaction basically consists of three parts: denaturation-annealing-extension:[0004]1) Denaturation of template DNA: After the template DNA is heated to 95°C or higher for a certain period of time, the template DNA double-stranded or the double-stranded DNA fragment formed by PCR ampl...

AI Technical Summary

Problems solved by technology

However, this scheme has several disadvantages: 1. Quantitative analysis cannot be performed; 2. An additional melting curve analysis is added, which increases the overall time of PCR detection; 3. The melting curve is usually not standard when testing unknown samples, making it difficult to judge the results

4. There are interferences such as primer dimers and non-specific amplification products

These shortcomings greatly limit the application of multiple melting curve PCR analysis in nucleic acid detection

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