Method of extracting, amplifying and detecting nucleic acid in single tube based on nano microsphere

Abstract

The invention discloses a method for detecting nucleic acid based on nano micro-bead nucleic acid extraction in a single tube, which takes nano-beads as the platform and completes extraction, amplification and detection of the nucleic acid in a single reaction. In this way, it can integrates extraction, amplification and detection to be a continuous process to ensure the extracted nucleic acid to totally participates amplification and detection, and it facilitates the high-throughput nucleic acid detection experiment. The technical program include: it releases the nucleic acid in the sample and mixes with nano-beads. The nucleic acid and nano-beads reacts to achieve nucleic acid -nano-bead complex if there is nucleic acid in the samples. The nucleic acid - nano-bead complex separates with other ingredients. The separated nucleic acid - nano-bead complex is directly used for the real-time nucleic acid amplification of the target nucleic acid detection system to make it clear whether there is specific nucleic acid sequences in the samples and determines their concentrations.

Description

technical field [0001] The present invention relates to the field of nucleic acid detection, more specifically, relates to a method of separating and purifying nucleic acid components in a sample, and then carrying out in vitro specific amplification and real-time detection thereof to clarify whether there is a target nucleic acid in the sample and to determine its concentration Methods. Background technique [0002] A problem often encountered in the fields of medicine and biology is to confirm the presence of target nucleic acids corresponding to a certain disease pathogen in a sample (such as blood from a blood donor or a sample of blood or body fluid from a patient) and to determine The concentration of the target nucleic acid. However, the target nucleic acid in the sample is usually in a small amount and exists in the sample to be tested together with various other nucleic acid sequences, so that the target nucleic acid in the sample cannot be directly detected. At p...

AI Technical Summary

Problems solved by technology

[0003] In fact, nucleic acid amplification detection is traditionally carried out sequentially and separately according to the above three steps (i.e. nucleic acid extraction, target nucleic acid specific amplification in vitro, and detection of amplification products), so there are some significant Disadvantages: ①Many open operations and easy contamination, including foreign nucleic acid contamination, sample cross-contamination, and nuclease contamination, etc.; ②Easy to cause nucleic acid loss and reduce detection sensitivity: including nucleic acid contamination caused by nucleic acid separation and purification operations Loss, and the nucleic acid recovered by separation and purification cannot be completely used for subsequent amplification detection; ③The measurement of "end point" directly affects the accuracy of quantification; ④The experiment period is long and manual operations are many, so the detection cost cannot be effectively reduced; ⑤Because Many influencing factors lead to poor repeatability of the experiment

[0010] The loss of the nucleic acid to be tested caused by the inability of the isolated and purified nucleic acid to be fully used for amplification and detection is particularly prominent for samples in which the target nucleic acid is only present in a small amount, such as the early detection of pathogenic infection in clinical practice; while elution and transfer The relatively time-consuming open operation is also the speed-limiting link that restricts the high-throughput of experiments