Establishment Of Specific Real-Time Quantitative Isothermal Amplification Assay For Aspergillus Fumigatus

ObjectiveWith the wide application of broad-spectrum antibiotics and immunosuppressive agents,the number of hematopoietic stem cells and organ transplantation patients increased,the incidence of invasive fungal disease(IFD)is increasing.The main pathogens that induce IFD clinically are Candida and Aspergillus.The new early diagnosis assay of IFD is in urgent need.Our subject is to establish a real-time quantitative RPA(qRPA)assay for fast detection of Aspergillus fumigatus by designing and screening the specific primers and probes from the target sequence,the Internal Transcription Space Domain(ITSD)of Aspergillus fumigatus.Methods? The Aspergillus fumigatus genomic DNA was extracted by the bead-deating method.? The primers and probes were designed on the basis of the ITSD of Aspergillus fumigatus with Primer Premier5.0 and BLAST,and the specific primers and probes were screened by PCR combined with agarose gel electrophoresis.?A50?l qRPA amplification detection system was established to detect the Aspergillus fumigatus genomic DNA.The detection system and conditions were optimized with CFX96 TouchTM real time fluorescent quantitative PCR instrument.? The clinical isolate strains were cloned using T-A cloning technique and identified by sequencing from biological company.Finally,the clinical isolate strains were detected by qRPA assay.Results?The bead-beating method is suitable for Aspergillus fumigatus genomic DNA extraction.?The primers f1/R1 are the specific primers of Aspergillus fumigatus and the probe P2 is the specific probe,and its amplified product length is 363 bp.?Theoptimized protocol of the specific qRPA detection system for Aspergillus fumigatus is below:(1)The optimized probe concentration is 1.6?M;(2)The optimized primers concentration are 5.2?M;(3)The optimized reaction temperature is 39?;(4)The amplification time is less than 15 min.?The qRPA detection performance evaluation is below:(1)No cross reaction with other fungi and human genomic DNA was observed;(2)The minimum detection limit is 31 copies/ml;?32 strains of fungi were isolated from the clinical samples,and qRPA could be used to detect the strains of Aspergillus fumigatus in 19 strains.ConclusionIn this paper,the results show that qRPA technology has advantages of specificity,sensitivity,easy to operation and less influencing factors.In addition,the constant temperature reaction conditions make it less demanding on the equipment.This study lay a foundation for application and development of molecular biological diagnostic techniques for IFD.