PCR detection method of multiple-target nucleic acid in single pipe and kit thereof

Abstract

The invention relates to a real-time PCR method of one or more kinds of target nucleic acid in multiple-detection samples in a signal PCR reaction vessel, which comprises the following steps: (1) mixing a sample, DNA polymerase, dNTP, internal contrast nucleic acid, a target nucleic acid primer pair, an internal contrast nucleic acid primer pair, one or more kinds of target nucleic acid probes and internal contrast nucleic acid probes in the signal PCR reaction vessel, wherein the probes are marked with fluorescent groups and quenching groups, and moreover, the fluoroscopic detection wavelengths of the fluorescent groups marked by various probes are different; (2)detecting fluorescent light with different wave lengths in real time to carry out the PCR reaction; (3) calculating a Ct value according to a fluorescent light detecting result to judge whether one or more kinds of target nucleic acid is stored in the sample; in additon, the invention also discloses a detection kit for the method and preparation, application, and the like of the corresponding detection kit. The multiple-detection method does not need special devices and can be widely used in the fields of laboratory investigation, food security, medicine, hygiene, and the like.

Description

technical field [0001] The present invention belongs to the technical field of nucleic acid detection, in particular, the present invention relates to a PCR method for multiplexed detection of target nucleic acid in a sample in a single PCR reaction vessel. In addition, the present invention also relates to the reagents involved in the above PCR method, such as internal controls, primers, probes, etc., as well as the detection kit used in the above method and the preparation and application of the corresponding detection kit. Background technique [0002] Polymerase chain reaction (PCR) is one of the most commonly used technologies in nucleic acid detection today. Real-time PCR technology uses real-time detectable markers such as fluorescent labels. It is used in nucleic acid detection, clinical diagnosis and molecular biology research. and other functions are becoming more and more important. This type of technology is currently applied in a wide range of fields and has be...

AI Technical Summary

Problems solved by technology

However, when traditional and improved real-time PCR (especially real-time fluorescent PCR) detects actual samples, even if only a single target nucleic acid is detected, it must face the interference of multiple nucleotides that may exist in the sample to the detection results; When performing multiple target nucleic acid detection in a single PCR reaction container (i.e. simultaneously detecting a variety of target nucleic acids), since primers and probes for detecting different target nucleic acids are introduced simultaneously, it is further necessary to face different primers and probes. the problem of mutual interference

In the face of these difficulties, the prior art often adopts complex operations and high-cost processing methods, and even some internal control monitoring systems cannot be introduced to eliminate false negatives, or the introduced internal control (molecules) themselves (usually nucleic acids) further Exacerbated the mutual interference with various target nucleic acids, primers and their probes

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