Development of Single-closed Tube Multiplex PCR Technology for Detection
10.13865/j.cnki.cjbmb.2025.04.1101
- VernacularTitle:单管闭管多重PCR检测技术的发展
- Author:
Ting-Ting HU
1
;
Yun-Long ZHANG
1
;
Bing-Jie ZOU
1
Author Information
1. 中国药科大学药物质量与安全预警教育部重点实验室,南京 210009
- Publication Type:Journal Article
- Keywords:
nucleic acid detection;
multiplex nucleic acid detection;
polymerase chain reaction(PCR);
single-tube reaction;
detection method
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(8):1115-1126
- CountryChina
- Language:Chinese
-
Abstract:
Nucleic acid detection technology has been widely applied in fields such as pathogen detection due to its characteristics of rapidity,sensitivity,and specificity.With the numerous nucleic acid markers related to diseases,the demand for multiplex nucleic acid detection is gradually increasing.Multiplex polymerase chain reaction(PCR)can simultaneously amplify multiple targets,but there are problems such as easy contamination when opening the tube during the analysis process after amplification and high technical requirements.With the continuous advancement of detection technology,a series of simple,re-liable single-tube multiplex PCR detection technologies that do not require opening the tube have emerged successively.A common technology is the single-closed tube multiplex PCR detection method based on fluorescent probes,which mainly uses different fluorescent labels to distinguish multiple targets.Com-bined with different specific enzymatic digestion reactions,it can achieve multiplex detection of rare tumor mutations and single nucleotide-specific genotyping.In addition,the monochromatic melting curve analysis method based on differences in melting temperatures enables parallel detection of multiple targets within a single fluorescence channel.When performed within multiple fluorescence channels,it is called the multicolor melting curve analysis method,which can increase the number of detected targets to doz-ens,greatly breaking through the limitation of the number of fluorescence channels on the multiplexity of detection.At the same time,the fluorescence coding method using different combinations of fluorescent labels also provides new ideas for single-closed tube multiplex PCR detection.These include encoding the sequence of signals generated by different fluorescent labels corresponding to the same target,using a combination of two fluorescent labels to identify specific targets,and controlling the amplitude of fluores-cent signals of different targets,all of which can also improve the multiplexity of detection.This article summarizes and prospects the research progress of single-closed tube multiplex PCR detection technology in recent years from multiple dimensions such as principles,applications,and the advantages and disad-vantages of the methods,providing valuable references for subsequent scientific research exploration and application.
