Simple and rapid detection Aspergillus fumigatus by loop-mediated isothermal amplification coupled with lateral flow biosensor assay.

Abstract

We have developed a new diagnostic technique, termed loop-mediated isothermal amplification coupled with lateral flow biosensor (LAMP-LFB), which has been successfully applied to the detection of Aspergillus fumigatus. A set of six LAMP primers was designed according to the A. fumigatus-specific anxC4 gene, which specifically recognized eight different regions of the target sequence. The LFB was employed for reporting the A. fumigatus-LAMP results, and the visual readouts were obtained within 2min. The strains of A. fumigatus species and non-A. fumigatus species were used to test the assay's sensitivity and examine the analytical specificity of the target assay. Optimal LAMP conditions were 66°C for 50min. The limit of detection is 100fg. No cross-reactions were obtained, and the specificity of LAMP-LFB assay was 100%. The whole process of the assay, including 20min of DNA preparation, 50min of constant temperature amplification, and 2min of detection by the sensor strip, took a total of 72min (less than 75min). Among 89 sputum specimens for clinical evaluation, 10 (11·23%) samples were A. fumigatus-positive by LAMP-LFB and traditional culture method, 9 (10·11%) samples were A. fumigatus-positive by PCR method. Compared with culture method, the diagnostic accuracy of LAMP-LFB method was 100%. The novel LAMP-LFB detection technology established in the current research is a rapid and reliable detection tool for A. fumigatus. This novel LAMP-LFB assay can quickly, specifically and sensitively detect A. fumigatus, thereby speeding up the detection process and increasing the detection rate. In addition, it can also be used as a new molecular method for detection of A. fumigatus in clinical and laboratory areas.