TRPCR (template-ready polymerase chain reaction) detection method of RNA (ribonucleic acid)

Abstract

The invention provides a TRPCR (template-ready polymerase chain reaction) detection method of RNA (ribonucleic acid). Compared with the traditional RT-PCR (reverse transcription-polymerase chain reaction) method, the method provided by the invention does not require RNA purification and extraction, omits reverse transcription reaction, and shortens the PCR pretreatment process from the original more than 3 hours to about 50 minutes. Thus, the method provided by the invention is simple to operate and quick and easy to implement, has high RNA detection sensitivity and specificity, and is suitable for the detection of RNA obtained by cracking a sample from any source.

Description

(1) Technical field [0001] The invention relates to a RNA preparation template PCR (Template-Ready PCR, TRPCR) detection method. (2) Background technology [0002] RNA is the carrier of genetic information transmission. RT-PCR (reverse transcription reverse transcription-polymerase chain reaction), refers to the method of combining reverse transcription (Reverse Transcription, RT) and PCR (Polymerase Chain Reaction) reactions. RT-PCR will use RNA as a template for cDNA Synthesis, or RT, combined with PCR provides a fast and sensitive method for analyzing gene expression. RT-PCR detection or quantitative analysis of RNA is more sensitive and easier to operate than other RNA analysis techniques including Northern blotting, RNase protection analysis, in situ hybridization and S1 nuclease analysis. Detection and analysis, detection and analysis of pathogens and other powerful means. [0003] The template for RT-PCR can be total RNA or poly(A)+RNA. RT requires reverse transcri...

AI Technical Summary

Problems solved by technology

The two-tube method is relatively common. In the two-tube method RT-PCR, each step can be carried out under the best conditions as much as possible. However, because the RT system contains PCR inhibitors, generally only about 5% of the RT product can be taken out for PCR. , which limits the sensitivity of the final assay, but is useful when using one sample to detect multiple target RNAs—since one RT product can be used separately for many PCR reactions

In one-tube RT-PCR, RT and PCR are carried out in one tube, and conditions optimized for both reverse transcription and PCR must be provided as much as possible. One-step RT-PCR is easy to operate when processing a large number of samples and helps to reduce residual Contamination, as there is no need to uncap the tube between cDNA synthesis and amplification. Successful optimization of the one-step method can result in higher sensitivity, down to 0.1 pg total RNA, because the entire cDNA sample is amplified – theory The upper sensitivity can be increased by 20 times compared with the two-tube method. However, it is difficult to control the conditions optimized for one-step RT-PCR, which often leads to serious non-specific amplification, and the cost is greatly increased.

[0004] Although RT-PCR has a high degree of promotion and popularity in the field of scientific research, there is still a lot of room for improvement in clinical detection applications. The main problems include: cumbersome steps and high requirements for operations, especially the two enzymatic reactions (RT-PCR The optimal reaction conditions of PCR and PCR are different, and they are easily affected by various inhibitory substances, non-specific amplification and false positives. Finally, the price of reverse transcriptase remains high, and the operation cost is very difficult. come down.

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