Establishment of a rapid diagnosis method for Candida glabrata based on the ITS2 gene using recombinase polymerase amplification combined with lateral flow strips

Abstract

Candida glabrata is the second or third most common Candida-associated species isolated from hospital-acquired infections, surpassing even C. albicans in some hospitals. With the rapid progression of the disease course of C. glabrata infections, there is an urgent need for a rapid and sensitive on-site assay for clinical diagnosis. Isothermal amplification is a recently developed method for rapid nucleic acid detection that is being increasingly used for on-site detection, especially recombinase polymerase amplification (RPA). RPA combined with lateral flow strips (LFS) can rapidly amplify and visually detect the target gene within 20 min. The whole detection process can be controlled within 30-60 min by rapid sample pre-treatment. In this study, RPA-LFS was used to amplify the internal transcribed spacer region 2 gene of C. glabrata. The primer-probe design was optimized by introducing base mismatches (probe modification of one base) to obtain a highly specific and sensitive primer-probe combination for clinical sample detection. RPA-LFS was performed on 23 common clinical pathogens to determine the specificity of the assay system. The RPA-LFS system specifically detected C. glabrata without cross-reaction with other fungi or bacteria. Gradient dilutions of the template were tested to explore the lower limit of detection of this detection system and to determine the sensitivity of the assay. The sensitivity was 10 CFU/µL, without interference from genomic DNA of other species. The RPA-LFS and qPCR assays were performed on 227 clinical samples to evaluate the detection performance of the RPA-LFS system. Eighty-five samples were identified as C. glabrata, representing a detection rate of 37.5%. The results were consistent with qPCR and conventional culture methods. The collective findings indicate a reliable molecular diagnostic method for the detection of C. glabrata, and to meet the urgent need for rapid, specific, sensitive, and portable clinical field-testing.

Keywords: Candida glabrata; ITS2; lateral flow strip; qPCR; recombinase polymerase amplification.