Universal primer for detecting aspergillus flavus and aspergillus fumigatus and real-time fluorescence tHDA (Thermophilic Helicase Dependent Amplification) kit

Abstract

The invention discloses a universal primer for detecting aspergillus flavus and aspergillus fumigatus and a real-time fluorescence tHDA (Thermophilic Helicase Dependent Amplification) kit. The universal primer can be used for simultaneously detecting the aspergillus fumigatus and the aspergillus flavus in an amplification manner and is simultaneously combined with a real-time fluorescence tHDA technology. Thus, compared with other common PCR (Polymerase Chain Reactions), according to the universal primer, the amplification can be carried out at a constant temperature, so that the processes of denaturation, annealing, extending and the like are omitted, i.e., the helicase amplification can be carried out by directly simulating a biology environment, which is favorable for simplification of equipment and popularization of the universal primer in grass roots. The detection limit of the real-time fluorescence tHDA kit can reach 104copies/ml, so that the real-time fluorescence tHDA kit is suitable for clinical applications. The real-time fluorescence tHDA kit can be used for detecting two types of aspergillus infections which are the most common in clinical, so that a large quantity of precious rescue time in clinical can be saved and the kit is low in cost and convenient to operate. Thus, the real-time fluorescence tHDA kit has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of in vitro diagnostic nucleic acid detection, and in particular relates to a fluorescent tHDA (thermostable helicase-dependent constant temperature amplification) kit for detecting infection of Aspergillus fumigatus and Aspergillus flavus in clinical samples. Background technique [0002] In recent years, with the increase of tumor patients, the widespread use of chemotherapy drugs, immunosuppressants and broad-spectrum antibiotics, especially in malignant hematological diseases and bone marrow transplantation, the incidence of invasive fungal infection has increased significantly, and Aspergillus infection It is an invasive fungal infection second only to Candida infection and is increasing year by year. Studies have shown that the mortality rate of pulmonary Aspergillus infection is 40%, and that of generalized Aspergillus infection is 90%. Aspergillus has become the primary pathogen in the autopsy cases of repeat...

AI Technical Summary

Problems solved by technology

Only using tHDA amplification technology to qualitatively or quantitatively analyze the final product cannot accurately quantify the initial template, while real-time fluorescent tHDA technology uses the change of fluorescence signal to monitor the change of the amount of amplification product in each cycle of HDA amplification reaction in real time. Quantitative analysis of the starting template can be carried out through the analysis of Ct value and its standard curve

However, the tHDA amplification technology is currently in its infancy in the world, although the existing tHDA amplification technology is used in the detection of Aspergillus (Wang Xiaoting. Research on Helicase-dependent DNA Constant Temperature Amplification Technology for Aspergillus flavus detection [D ]. Fujian Medical University, 2012.), but its sensitivity is low, and the starting template cannot be quantitatively analyzed. At present, no real-time fluorescent tHDA technology has been applied to the detection of Aspergillus at home and abroad.

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