Method, probe and kit for detecting multiple target nucleic acid sequences to be detected through single tube

Abstract

The invention provides a method, a probe and a kit for detecting multiple target nucleic acid sequences to be detected through a single tube. The method comprises the steps of designing specific upstream and downstream primers and a probe against each target nucleic acid sequence to be detected, and controlling a fluorescence product melting point Tm value of each target nucleic acid sequence to be detected, wherein the probe contains at least one RNA base; labeling a fluorescence group on a probe base close to one side of a 5' terminal of the probe on the left side of the RNA base; and labeling a quenching group on a probe base close to one side of a 3' terminal of the probe on the RNA base. According to the invention, through semi-nested amplification of reaction primers and the amplification of the probe and the primers after enzyme digestion of the RNA base(s), the detection specificity and the sensitivity are improved; through adoption of RNaseH enzyme digestion, the primers withfluorescence labels are obtained, and specific products are directly subjected to melting curve analysis, thus realizing excellent specificity.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for detecting multiple target nucleic acid sequences to be detected in a single tube, a probe and a kit thereof. Background technique [0002] Traditional real-time fluorescent PCR technology adds different fluorescent groups to the PCR reaction system, and uses the accumulation of fluorescent signals to monitor the entire PCR process in real time. It has the advantages of high sensitivity, high specificity, effective solution to the problem of PCR contamination, and rapidity. It is currently a widely used method for nucleic acid detection. However, due to the limitation of the channels of fluorescent PCR instruments on the market, a single tube can only detect 4-5 targets at most, which cannot meet the clinical needs of simultaneous screening of multiple pathogens or genes related to a certain syndrome. [0003] At present, the common pathogenic microorganism...

AI Technical Summary

Problems solved by technology

However, there are still some problems to be solved in this technology: the massive data generated by sequencing requires professional personnel to analyze; the time and cost of sequencing still restrict its wide application in clinical practice

[0007] In view of the current clinical needs for simple, fast, high sensitivity, good specificity and multi-target detection technology, and the existing technology has its limitations and cannot fully meet the clinical needs, we urgently need to develop a fast, accurate and low-cost multiple detection technology

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