Kit for one-step nucleic acid quantitative RT-PCR detection on Xinjiang hemorrhagic fever virus

Abstract

The invention provides a kit for one-step nucleic acid quantitative RT-PCR detection on a Xinjiang hemorrhagic fever virus.The kit comprises RT-PCR reaction liquid, a RT-PCR enzyme mixture, XHFV nucleic acid quantitative primers and probes, an internal reference, negative control, critical positive control, strong positive control and calibrators (1-5).According to the kit, a one-step RT-PCR reaction can be directly performed on extracted XHFV RNA, fluorescent quantitative detection is performed on XHFV RNA in the samples, an internal reference gene sequence is taken as internal control, and contamination is prevented through a UNG enzyme; the kit is simple in one-step amplification method, short in procedure, easy and convenient to operate, capable of preventing contamination, high in specificity of a detection result and sensitivity, clear in result and high in credibility and can be applied to nucleic acid quantitative detection on the Xinjiang hemorrhagic fever virus in serum.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to a biological detection technology, in particular to a kit for fluorescent quantitative detection of Xinjiang hemorrhagic fever virus nucleic acid. Background technique [0002] Crimean Congo hemorrhagic fever (CCHF) was discovered in Crimea of ​​the former Soviet Union in 1944. It is caused by Crimean Congo hemorrhagic fever virus (CCHFV). The average case fatality rate of severe infectious diseases in southern Xinjiang of China, northern Russia, Middle East, southern Eurasia and African Sahara is between 10% and 50%. China's CCHF was first discovered in southern Xinjiang in 1965, and antibodies were also detected in northern Xinjiang later, so it was called Xinjiang hemorrhagic fever (XHF). [0003] Xinjiang hemorrhagic fever virus (XHFV) is isolated from the blood of patients with hemorrhagic fever in Tarim area, Xinjiang, the liver, spleen, kidney, lymph nodes of corpses, and h...

AI Technical Summary

Problems solved by technology

At present, there is no XHFV nucleic acid quantitative detection reagent on the market at home and abroad; the development of this kit can fill the gap in technology and products in this area

[0018] At the same time, in order to avoid false positives in the reaction results, using the anti-pollution characteristics of UNG enzyme, dUTP is used instead of dTTP in the PCR amplification reaction, so that only dUTP is contained in the amplified fragment; and dUTP makes the polluted amplicon amplified The target DNA is easily degraded by UNG enzyme: this dUTP-containing PCR product is incubated with UNG enzyme, because UDG enzyme can cleave the N-glycosyl bond between the uracil base and the sugar-phosphate backbone, and can be single-stranded or double-stranded Elimination of dUTP in DNA prevents the extension of TaqDNA polymerase and thus loses the ability to be reamplified

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