Establishment and application of loop-mediated isothermal amplification for detecting clinical isolates of Aspergillus spp

Abstract

Objective To establish a method for detecting Aspergillus spp.by Loop-mediated isothermal amplification. Methods Aspergillus spp.specific primers were designed from the relative conservation region of the published sequence of 28S rRNA genes of A.fumigatus (GenBank accession number AY660917),A.terreus (GenBank accession number AF454183),A.flavus (GenBank accession number AF454158),and A.niger (GenBank accession number AF454169).Genomic DNA were extracted from Aspergillus standard strains,clinical control strains and clinical samples,and amplified by LAMP.The amplified results could be read with the naked eye by the coloring effect of fluorescent nucleotide dye without the DNA electrophoresis.Approximately 103 each Aspergillus conidia suspension was added to the serum from healthy volunteers,and detected these simulative clinical samples by LAMP assay. Results The LAMP and PCR assays obtained positive results for all four Aspergillus species and 8 simulative clinical samples (double samples for each Aspergillus species) including 103 conidia,but negative in the remaining 15 non-Aspergillus species,human total blood genomic DNA,30 clinical serum samples infected with non-Aspergillus and 10 healthy volunteers.The LAMP assay had a minimum detection of 0.05-0.5pg,by means of detect different levels of 500,50,5,0.5,0.05 pg template in each reaction tube. Conclusion The results confirm that LAMP is a simple,rapid,sensitive and specific method,and can be used for detection of Aspergillus strains in clinical and environmental specimens.（Chin J Lab Med,2014,37:140-143） Key words: Aspergillus; Nucleic acid amplification techniques; Sensitivity and specificity