Development and Application of a Multiple Cross Displacement Amplification Combined With Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Candida tropicalis.

Yu Wang,Xue Zhao,Xiaomin Tang,Jinzhi Cheng,Shijun Li,Xu Chen,Honglan Yu

doi:10.3389/fmicb.2021.681488

Abstract

Candida tropicalis is an increasingly opportunistic pathogen that causes serious invasive candidiasis threatening a patient’s life. Traditional methods to detect C. tropicalis infection depends on time-consuming, culture-based gold-standard methods. So, we sought to establish a new method that could detect target pathogens quickly, accurately, and straightforwardly. Herein, a combination of multiple cross displacement amplification (MCDA) and lateral flow biosensors (LFB) was employed to detect C. tropicalis. In the MCDA system, 10 primers were designed to identify the specific genes of C. tropicalis and amplify the genes in an isothermal amplification device. Then, MCDA amplification reaction products could be identified visibly by color change, and all the amplification products would be tested by LFB with no special equipment. The results demonstrated that the optimal reaction condition of C. tropicalis-MCDA assay was 64°C within 30 min, and only 10 fg DNA was required in each reaction. No cross-reaction was found between C. tropicalis strains and non-C. tropicalis strains. For 300 sputum samples, the results showed that MCDA-LFB assay could rapidly and successfully detect all of the C. tropicalis-positive (28/300) samples detected by the gold-standard method. The entire procedure, including specimen processing (40 min), isothermal reaction (30 min) and result reporting (within 2 min), could be completed within 75 min. Briefly, the study results demonstrated that the detection ability of C. tropicalis-MCDA-LFB assay was better than culture methods with more simplicity, rapidity, sensitivity and specificity. Hence, MCDA-LFB strategy is an effective tool to rapidly detect C. tropicalis in clinical samples, especially in resource-poor areas.

Highlights

Invasive candidiasis is a well-known life-threatening disease and a major issue for a medical institution, leading to significant morbidity, mortality, and huge extra hospital costs (Fuchs et al, 2019; Jenks et al, 2020)
To confirm the effectiveness of the multiple cross displacement amplification (MCDA) primers we selected (Figure 1 and Table 1), the amplification products were monitored by two different methods, including colorimetric indicator and lateral flow biosensor (LFB)
The results showed that the positive results were observed when the nucleic acid from C. tropicalis, but not with C. albicans, Klebsiella pneumoniae, and the blank control (Figures 2A,B)

Summary

Introduction

Invasive candidiasis is a well-known life-threatening disease and a major issue for a medical institution, leading to significant morbidity, mortality, and huge extra hospital costs (Fuchs et al, 2019; Jenks et al, 2020). Some previous studies indicated that invasive candidiasis caused by C. tropicalis have higher mortality compared to those caused by other non-tropicalis Candida species (Wang et al, 2016a; Fan et al, 2017). When patients with low immunity, especially those with neutropenia, leukemia, tumors, and bone marrow transplants are susceptible to C. tropicalis, this causes various clinically relevant infections, including candidiasis and systemic disseminated infections resulting in significant morbidity and mortality (Wang et al, 2016b; Horváth et al, 2020)

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Discussion

Conclusion