Establishment and Application of Multiple Cross Displacement Amplification Coupled With Lateral Flow Biosensor (MCDA-LFB) for Visual and Rapid Detection of Candida albicans in Clinical Samples.

Fan Zhao,Lina Niu,Shoukui Hu,Linlin Yan,Xiaoxue Zhu,Chunmei Wang,Jing Wang,Fengzhi Zheng,Lei Wu,Naishu Gao,Jinqing Nong

doi:10.3389/fcimb.2019.00102

Abstract

Candida albicans is an opportunistic pathogenic yeast that predominantly causes invasive candidiasis. The conventional diagnosis of C. albicans infection depends on time-consuming, culture-based gold-standard methods. Here, a multiple cross displacement amplification (MCDA) assay, combined with a gold nanoparticle-based lateral flow biosensor (LFB) visualization method, was developed for the rapid detection of C. albicans. The internal transcribed spacer II, a region between 5.8 and 28 S fungal ribosomal DNA, is a C. albicans species-specific sequence that was used as the MCDA assay target. As an isothermal amplification method, the MCDA reaction with optimized conditions could be completed within only 40 min at a constant temperature (64°C). Then, the amplification reaction products could be visibly detected by a LFB without special equipment. The developed MCDA-LFB assay for C. albicans detection was a specific and accurate method, and could distinguish C. albicans from other pathogens. Just 200 fg of genomic DNA template from pure cultures of C. albicans could be detected using the MCDA-LFB method. The limit of detection (LOD) of the new method was more sensitive than that of both qPCR and loop-mediated isothermal amplification (LAMP). Of 240 clinical sputum samples, all of the C. albicans-positive (87/240) samples identified by the gold-standard method were successfully detected by the MCDA-LFB assay. Moreover, the true positive rate of the newly developed assay was not only higher than that of qPCR (100 vs. 86.2%), but also higher than that of LAMP (100 vs. 94.3%). Thus, the MCDA-LFB assay might be a simple, specific, and sensitive method for the rapid diagnosis of C. albicans in clinical samples.

Highlights

Candida albicans is a member of the human gut flora and it is an opportunistic pathogenic yeast (Gow and Yadav, 2017)
Several non-culture methods have been developed for C. albicans detection, including mass spectrometry (Zehm et al, 2012), immunoassay (Gunasekera et al, 2015), polymerase chain reaction (PCR) (Vahidnia et al, 2015), real-time PCR (Maaroufi et al, 2003; Kasai et al, 2006), polymerase spiral reaction (PSR) (Jiang et al, 2016), and loop-mediated isothermal amplification (LAMP) (Noguchi et al, 2017)
We aimed to establish a multiple cross displacement amplification (MCDA)-lateral flow biosensor (LFB) assay for C. albicans detection via the species-specific sequence from ITS Internal Transcribed Spacer II (II) region with DNA template from pure cultures of the reference strain, following it, we applied the assay to clinical samples

Summary

Introduction

Candida albicans is a member of the human gut flora and it is an opportunistic pathogenic yeast (Gow and Yadav, 2017). The current gold-standard method for C. albicans detection is based on phenotyping, including culture, microscopic examination, and biochemical identification. It typically requires more than 2 days for growth and identification, which is limited by slow turn-around times; positive identification may occur late in the course of infection. Compared with the gold-standard culture method, these tests for C. albicans identification can save considerable time. Such assays depend on technical expertise or complex equipment, which may not be available in some settings

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Results