Novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers and detection method

Abstract

The invention provides novel high-sensitivity respiratory virus nucleic acid NASBA (nucleic acid sequence based amplification) primers. A respiratory virus conservative area is selected, and a promoter sequence capable of being recognized by T7 RNA (ribose nucleic acid) polymerase is formed at the 5' terminal of one of the synthesized primers. The invention further provides a novel high-sensitivity respiratory virus nucleic acid NASBA detection method. T7 RNA polymerase promoter sequences are added to the 5' terminals of the two primers complementary to two ends of a respiratory virus RNA sequence, so that when the primers enter a circulation phase, RNA sequences [RNA(+)] consistent with a template sequence can be synthesized, RNA sequences [RNA(-)] complementary to a template RNA sequence can be synthesized, and the detection sensitivity of viral nucleic acid is greatly improved; a throat swab sample of a patient can be directly used for amplification, a reverse transcription process is not required, and the operation time is saved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a gene amplification technology for respiratory virus detection, and more specifically to establish a high-sensitivity NASBA amplification technology for respiratory virus nucleic acid. Specifically, it includes a pair of respiratory virus-specific amplification primers, a NASBA amplification reaction system and a NASBA product detection system. Background technique [0002] Acute respiratory infection is one of the main causes of human morbidity and death. There are many types of pathogens that cause infection, including bacteria, viruses, mycoplasma and chlamydia, etc., especially viruses. Regardless of the etiology, the clinical symptoms and signs of respiratory tract infections are similar, while infections caused by different types of pathogens have completely different treatment methods, and the curative effects and course of the disease are also different. [0003] R...

AI Technical Summary

Problems solved by technology

However, this method needs to first reverse-transcribe the viral RNA into cDNA, and then perform the amplification reaction of the specific nucleic acid sequence, which increases the chance of contamination and limits the wide application of this technology in clinical testing.

The fourth category is gene chips, next-generation sequencing technology, and mass spectrometry. Although these advanced detection technologies have greatly improved sensitivity, they are time-consuming or expensive, and have many scientific research uses. They have not yet been widely used in clinical pathogen detection. application

It can be seen that the classic NASBA technology coupled with enzyme-labeled (fluorescent) probe hybridization method can only recognize the RNA sequence consistent with the template strand, and the detection process must be coupled with enzyme-labeled (fluorescent) probe color technology, Increased the operational complexity of the method and the need for color development equipment, etc.

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