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

Abstract

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. 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 LFB 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 min. The limit of detection is 100 fg. No cross-reactions were obtained, and the specificity of LAMP-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 50 min of constant temperature amplification, and 2 min of detection by the sensor strip, took a total of 72 min (less than 75 min). Among 89 sputum specimens for clinical evaluation, 10 (11Á23%) samples were A. fumigatus-positive by LAMP-LFB and traditional culture method, 9 (10Á11%) 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.

Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens

“…LAMP offers a rapid and high throughput alternative for detection of several types of disease-causing fungi viz. Candida albicans, Cryptococcus neoformans and Mucor racemosus ( Nakayama et al., 2017 ) , Trichosporonasahii ( Zhou et al., 2015 ), Aspergillus fumigatus ( Jiang et al., 2021 ) , Pythium insidiosum ( Htun et al., 2020 ) , etc. These studies have shown the requirement of a very low amount of DNA for LAMP assay which shows the high sensitivity of this method.…”

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

“…Lateral flow strips from different manufacturers-Millenia [197,234,235], Foresite Diagnostics [230,233,236], Biohelix [229], and AMODIA Bioservice [237]-were implemented for the detection of labelled amplicons. Additionally, homemade dipsticks utilizing NP-streptavidin conjugate were used [237].…”

“…Lateral flow strips from different manufacturers-Millenia [197,234,235], Foresite Diagnostics [230,233,236], Biohelix [229], and AMODIA Bioservice [237]-were implemented for the detection of labelled amplicons. Additionally, homemade dipsticks utilizing NP-streptavidin conjugate were used [237]. Different approaches were designed to obtain labelled products, including FIP/BIP-labelled primers [229,236], FIP-/loop-labelled primers [237], loop-labelled primers [230,233], and biotin-labelled probes for hybridization of FITC-labelled amplicon [197,234,235].…”

“…Additionally, homemade dipsticks utilizing NP-streptavidin conjugate were used [237]. Different approaches were designed to obtain labelled products, including FIP/BIP-labelled primers [229,236], FIP-/loop-labelled primers [237], loop-labelled primers [230,233], and biotin-labelled probes for hybridization of FITC-labelled amplicon [197,234,235]. The overall analysis comprised extraction of DNA, amplification, optional hybridization, and LFA.…”

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

“…Therefore, isothermal amplification assays showed great potential in scientific and clinical research [ 26 ]. Loop-mediated isothermal amplification (LAMP) is one of the well-recognized isothermal amplification techniques applied in clinical diagnosis [ 27 , 28 ]. But LAMP needs specific design of 2 to 3 pairs of primers and the processes of primer design and selection are tedious [ 29 , 30 ].…”