Rapid Detection Of Aspergillus Fumigatus With Recombinase Polymerase Amplification Assay

[Objective]To screene specific primers targeting the ITSD sequence of Aspergillus fumigatus, then estabilish a new rapid detection method of Aspergillus fumigatus with Recombinase Polymerase Amplification Assays,and optimize.[method](1) Designed three forward and three reverse primers targeting variant region ITSD sequence of Aspergillus fumigatuss with primer 5.0 software, combined these forward and reverse primers, then screened specific primer of Aspergillus fumigatus with primer-blast software from these combination primers.(2) Eextracted genome DNA of standard strain of Aspergillus fumigatus by bead-beating method.(3) Estabilished a new method for detection of Aspergillus fumigatus by optimizing the RPA conditions of detection Aspergillus fumigatus,including primer length,production length,amplification time and amplification temperature.(4)Assessed sensitivity of RPA by detecting different concentrations of Aspergillus fumigatus DNA, and assessed spercificity of RPA by cross-detection of defferent strains of Aspergillus.(5)Assessed the clinical detection performance by detecting 21 clinical isolated strains of Aspergillus which is human pathogenic strains.(6)Compared methodology of RPA to PCR through simultaneously carrying on experiments of sensitivity, specificity,clinical isolated strains detection of with this two technology.[Results](1) The forwared primer of screened screened specific primer of Aspergillus fumigatus is located upstream of ITSD1, and the reverse primer is located downstream of ITSD2. The combination primers can amplify an amplicon with size of 489bp, the sequence of which as following: Forwared:5’-GGTCCAACCTCCCACCCGTGTCTATC-3’; Reverse:5’-TTAGAAAAATAAAGTTGGGTGTCGGC-3’(2) he genomic DNA of Aspergillus fumigatus could meet the requirement of amplification in purity and auantitv obtained by bead-beatine extraction method, which is simple and fast.(3) The optimization reaction conditions of RPA for detection of Aspergillus fumigatus as following: primer length range:18-32bp; amplicon length range:200-700bp; amplification temperature:37-42℃ amplification time:15-40min(4) Specificity and sensitivity of RPA:experiment results of cross-detection of defferent Aspergillus showed:RPA only detects Aspergillus fumigatus and shows no cross-detection of, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, Rhodotorula sinensis, has the same sensitivity of detection limit of 100pg/μl DNA by gel electrophoresis;(5) Results of RPA detection 21 clinical isolated Aspergillus strains which is human pathogenic showed:RPA could detect the 18 clinical isolated strains of Aspergillus fumigatus fumigatus, and could not detect 18 clinical isolated strains of Aspergillus flavus.(6) Compared methodology of RPA to PCR in the detection of Aspergillus fumigatus, we found RPA had the same high sensitivity and strong specificity as PCR.[Conclusion]This study screened specific primers targeting ITS1-ITS2 DNA sequence of the Aspergillus fumigatus by Primer-Blast. Under the optimization reaction condition, the method of RPA for detection of Aspergillus fumigatus with screened specific primers can effectively amplify the DNA of Aspergillus fumigatus under low isothermal condition, which has the characteristics of simple, fast, low cost, and has the same sensitivity and specificity of with the PCR technology, is suitable for rapid detection and identification of Aspergillus fumigatus.