Nucleic acid amplification detection method and detection kit for distinguishing DNA from corresponding RNA

Abstract

The invention belongs to the technical field of medical detection, in particular relates to a nucleic acid amplification detection method for distinguishing DNA from correspondingly transcribed RNA and provides a corresponding detection kit. The kit comprises pyrolysis solution, nucleic acid extracting solution, lysozyme solution, reverse transcription buffer solution, a reverse transcriptase system, tuberculosis DNA PCR reaction solution, tuberculosis RNA PCR reaction solution and the like, wherein selected primers aim at a section of ITS sequence or IS6110 sequence and a section of 16S rRNA sequence on ribosomal RNA respectively; and a probe is Tagman MGB probe preferably and labeled by different fluorescein. The kit comprises a glycosidase (UNG) component, can judge the relative 16S rRNA content of each bacterium and is used for judging the growth condition of mycobacterium tuberculosis in a sample so as to monitor infection state. The kit is simple, convenient and fast, can specifically detect the RNA and DNA and provides more comprehensive information for clinical use.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a nucleic acid amplification detection method and a detection kit for distinguishing DNA from corresponding RNA. Background technique [0002] Tuberculosis is a chronic wasting infectious disease that seriously affects human health. Due to factors such as immigration, drug abuse, co-infection of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis, multidrug resistance of Mycobacterium tuberculosis, and zoonotic infectious diseases, there is a new infection every second in the world, and new cases occur every year. There are 8.5-10 million tuberculosis patients, 3 million people die from tuberculosis, and the new incidence rate is increasing year by year. Currently, about 1-2 billion people are infected with tuberculosis worldwide. This poses new challenges to global TB control. The dire situation with tuberculosis has prompted Global Fund ag...

AI Technical Summary

Problems solved by technology

The main problem is that the equipment and reagents are expensive, the detection time is still long, and the reliability of the results is still controversial compared with the traditional culture method, etc., and it is difficult to use it as a rapid detection

(6) Phage biological amplification method: high sensitivity, up to 100 bacteria / ml, but compared with bacterial culture, the test conditions are more stringent and the detection rate is low, so the current evaluation is not high

However, there is no reagent for simultaneously detecting its DNA and RNA in the existing tuberculosis diagnosis. The reason is that there is a lack of a nucleic acid amplification method that can specifically detect its DNA and RNA simultaneously, such as the PCR method. Amplification of RNA requires a reverse transcription (RT) process to convert RNA into DNA and then amplify. The corresponding DNA and RNA cannot be distinguished. For example, when 16Sr RNA is detected by RT-PCR, the corresponding 16Sr DNA is also detected. Efficiently amplified, indistinguishable from DNA or RNA detected

Another example is the TMA method. Due to the use of isothermal amplification technology, the temperature is low, the chromosomal DNA is not denatured, and the single-stranded RNA is involved in the amplification. Therefore, it cannot be directly used for DNA detection unless a step of "high temperature denaturation" is added before amplification. steps; in addition, even with the addition of a heat denaturation step, it is impossible to distinguish DNA from RNA

Nucleic acid reagents for the single detection of tuberculosis DNA or RNA cannot give more information than sputum culture. Experts believe that nucleic acid diagnosis has not achieved the expected results. Most scholars believe that its diagnostic accuracy is not as good as bacterial culture, and some scholars do not even Recognize the results of fluorescent PCR

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