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.

Figure 1

Screening of primer–probe combinations. (A) RPA results for two different primer sets of the ITS2 gene. The name of each primer group is shown at the top of each lane. NTC indicates the no-template control for the corresponding primer pair. All reactions were performed at 37°C for 20 min. These images represent the results of three independent experiments. (B) Pairwise analysis and sequence modification of the primer–probe set were used to detect the ITS2 gene using Primer Premier 5 software, and the associated DNA base substitutions for the probes and primers. The DNA strands are shown as horizontal lines and the matching bases are shown as vertical lines. Molecular markers are listed below in Figure (B). (C) RPA-LFS assays for the validity of primer–probe sets. The name of each primer set is shown at the top of each lane. NTC denotes no-template control for the corresponding primer pair. The positions of the test and control lines are shown on the right. All reactions were performed at 37°C for 20 min. These images represent the results of three independent experiments.

Figure 2

Determination of the LOD of C glabrata RPA-LFS. (A) The LOD of the C glabrata RPA-LFS assay system was established by using primer–probe set F4/R2/P using genomic DNA of C glabrata and serial dilutions of 10⁰ to 10⁶ CFU for each reaction. The picture shows the results of RPA-LFS with the number of templates shown at the top of the bar graph. (B) The picture shows the results of the RPA-LFS assay using primer–probe set F4/R2/P and 10⁵ CFU of C albicans as interference.

Figure 3

Validation of primer pair F4/R2/P specificity against C. glabrata. (A) #1–#20 are the 20 C. glabrata isolated from clinical samples. NTC denotes no-template control. The positions of the test and control lines are marked on the right side of the bar graph. Reactions were performed at 37°C for 20 min. Images represent the results of three independent experiments.

Figure 4

Specificity of F4/R2/P. C. glabrata ATCC 15126 was used as a positive control against other pathogenic bacteria tested, and species names are indicated at the top of each strip. NTC indicates no-template control. The positions of the test and control lines are marked on the right side of the bars. Reactions were performed at 37°C for 20 min. Images represent the results of three independent experiments.