Method for detecting one or more target nucleic acid sequences to be tested by single tube and kit thereof

Abstract

The invention provides a method for detecting one or more target nucleic acid sequences to be tested by a single tube and a kit thereof. The method comprises the steps of designing a specific isothermal amplification primer and a fluorescent probe RNHP for each target nucleic acid sequence to be tested, and labeling each of target nucleic acid sequence fluorescent probes RNHP to be tested with different fluorescence; in the presence of ribonuclease RNaseH, isothermally amplifying each target nucleic acid sequence to be tested by the action of polymerase; forming hybridization chain products labeled with the different fluorescence by the different target nucleic acid sequences to be detected, thereby achieving multiplex detection of the plurality of target nucleic acid sequences to be tested. Based on an isothermal amplification technique, the method can achieve real-time multiplex isothermal detection by introducing a special modified primer RNHP and being combined with RNaseH2.

Description

Technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a method for detecting one or more target nucleic acid sequences to be tested in a single tube and a kit thereof. Background technique [0002] The isothermal nucleic acid amplification method, also known as the isothermal nucleic acid amplification method, is a rapidly developing nucleic acid amplification method since the beginning of the 21st century. Unlike PCR, which requires variable temperature and thermal denaturation to unlock double-stranded amplification, the isothermal nucleic acid amplification method is to rapidly amplify nucleic acid by adding different nucleic acid polymerases and specific primers at a constant temperature. . Isothermal amplification technology can basically achieve the detection of target nucleic acid within 1 hour, and some can even achieve detection within 20 minutes. [0003] The determination methods of isothermal amplification r...

AI Technical Summary

Problems solved by technology

[0003] The judgment methods of isothermal amplification results can basically be divided into three categories: 1. Run agarose gel electrophoresis and observe the gel map; this kind of result judgment method is time-consuming and laborious It is not conducive to product promotion. Generally, it may only be used in the product development stage, but it is basically not used in the actual nucleic acid isothermal detection products to judge the results.

2. Visual observation method to observe the turbidity or color change of the reaction tube before and after amplification; this kind of judgment method is simple and easy, and does not require high requirements for operators. It is a common result judgment method in the market, but its existence cannot distinguish specific Defects of amplification and non-specific amplification and the inability to do multiple amplification

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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