Fluorescent PCR detection system, kit and detection method for joint detection of various respiratory bacteria

Abstract

The invention discloses a fluorescent PCR (Polymerase Chain Reaction) detection system for joint detection on multiple respiratory tract bacteria and a kit and a detection method. The fluorescent PCRdetection system comprises a non-fluorescent amplification primer system, a detection probe system and a fluorescent amplification primer system. Aiming at the bottleneck problem that fluorescent PCRdetection is limited by detection channels, the fluorescent PCR detection system is based on two technical principles that a fluorescent signal is generated from a Taq enzyme hydrolysis fluorescent probe and a fluorescent signal can be also generated from product hybridization after a fluorescent primer is amplified, one target point in a single-color fluorescent channel is detected by using the fluorescent primer, another target point is detected in a mode that a fluorescent primer melting curve of direct amplification is detected by using the fluorescent probe, and detection and analysis ontwo target points are simultaneously implemented in the single-color fluorescent channel, so that joint detection on multiple respiratory tract bacteria can be carried out. The fluorescent PCR detection system, the kit and the detection method have the characteristics of being high in flux, low in cost and short in time.

Description

technical field [0001] The invention relates to the field of biochemical detection, in particular to a fluorescent PCR detection system, kit and detection method for joint detection of various respiratory bacteria. Background technique [0002] According to statistics released by the World Health Organization in 2013, among the top ten causes of death in the world, lower respiratory tract infection accounted for 5.9%, ranking third, and chronic obstructive pulmonary disease (accounting for 5.4%) ranked fourth. Also more relevant with respiratory tract infection. Pneumonia is one of the leading causes of death from infectious diseases worldwide, and it is the number one cause of death among children. According to the incomplete statistical analysis of the Chinese Center for Disease Control and Prevention in 2011, 184 to 223 children under the age of 5 in mainland China died of pneumonia for every 100,000 people. Secondary bacterial infection due to decreased immunity is the...

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Problems solved by technology

For example, respiratory tract infection can be caused by 30-40 common pathogens. If only one indicator is detected, the clinical sensitivity will be greatly reduced no matter how high the detection sensitivity AS is. Therefore, multiplex PCR technology is getting more and more attention in the molecular detection industry. The problem to be solved is to allow molecular detection to see not only the problems on the spot but also the things on the surface, thereby increasing the sensitivity of biochemical identification

[0005] However, although the in vitro detection industry has relied on real-time fluorescent PCR technology as a molecular detection tool for more than 20 years, with the development of the detection industry, the detection of multi-genes and multi-pathogens has become more and more urgent. Many attempts have been made, but no significant progress has been made. It is impossible to use a limited number of detection channels in a reaction tube to increase the throughput of target detection. Faced with the multi-target detection of more and more complicated complex pathogen infections Due to its high throughput, high efficiency, low cost and less time-consuming characteristics, multiplex PCR amplification has become one of the most ideal and fastest molecular detection methods. However, when performing multiplex PCR amplification, experiments have to Facing new problems, such as adding a pair of primers will increase the challenge of PCR reaction system design and optimization

[0006] Although fluorescent quantitative PCR has developed rapidly in the past two decades and has penetrated into all walks of life in the development of biotechnology, but with the increasingly high requirements of biological detection technology, this technology has also exposed many problems that need to be solved urgently. For example, there are 4-5 detection channels in the existing common fluorescent quantitative instruments, and the number of target genes detected by a single tube is generally 4-5, which has also become a bottleneck for multiplex real-time quantitative PCR detection

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