Simple and rapid detection Aspergillus fumigatus by loop-mediated isothermal amplification coupled with lateral flow biosensor assay
J Appl Microbiol. 2021 Mar 31. doi: 10.1111/jam.15092. Online ahead of print.
AIMS: 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. fumigatus).
MATERIAL AND METHODS: 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 lateral flow biosensor was employed for reporting the A. fumigatus-LAMP results, and the visual readouts were obtained within 2 min. 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 50 mins. The limit of detection (LoD) is 100fg. No cross-reactions were obtained and the specificity of LAMP-LFB assay was 100%. The whole process of the assay, including 20 mins of DNA preparation, 50 mins of constant temperature amplification, and 2 mins of detection by the sensor strip, took a total of 72 mins (less than 75 mins). 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%.
CONCLUSIONS: The novel LAMP-LFB detection technology established in the current research is a rapid and reliable detection tool for A. fumigatus.
SIGNIFICANCE AND IMPACT OF THE STUDY: 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.