The Journal of Practical Medicine >

2024 , Vol. 40 >Issue 19: 2738 - 2746

DOI: https://doi.org/10.3969/j.issn.1006-5725.2024.19.013

Medical Examination and Clinical Diagnosis

Rapid diagnosis of invasive candidiasis by droplet digital PCR

  • Zhijie HE ,
  • Weichao LI ,
  • Minghui HE ,
  • Xiaotong CHEN ,
  • Zhao LIN ,
  • Yaowei. ZHI
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  • *.Faculty of Rehabilitaion Medicine，Guangzhou Xinhua University，Guangzhou 510520，Guangdong，China

Received date: 2024-05-31

  Online published: 2024-10-22

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Abstract

Objective To establish a rapid detection method for invasive candidiasis based on droplet digital polymerase chain reaction （ddPCR）. Methods We developed an assay system using a microtitre-based digital PCR platform and designed primer probes specific for four Candida species， namely Candida albicans， Candida smoothii， Candida near-smoothii， and Candida tropicalis. （1） The Limit of Blank （LOB） range and positive judgment value were determined by analyzing No Template Control （NTC） samples. （2） The Limit of Detection （LOD） range was determined by diluting positive samples with 10 replicate extractions at each concentration gradient. （3） The Linear Limit of Quantitation （LOQ） range was determined by repetitive testing of diluted samples. （4） The linear range limit was determined through gradient dilution of the positive samples. （5） The coefficient of variation （CV）， calculated from the logarithmic values of the resultant concentrations， was assessed by extracting and testing positive samples in 12 repetitions at both high and low concentrations. （6） Method reliability was evaluated by calculating the CV from the logarithmic values of the resultant concentrations obtained from clinical samples with fungal culture results. Results The ddPCR assay detected Candida LOB at a range of 0 ~ 81 copies/mL， with a positive threshold set at ≥ 3 positive microdroplets. The LOD and LOQ were determined to be 3 × 102 copies/mL. The linear range for detecting different concentration gradients was found to be between 3 × 102 and 3 × 107 copies/mL， with high correlation coefficients observed for Candida albicans （R2 = 0.999 5）， Candida smoothii （R2 = 0.998 9）， Candida near-smoothii （R2 = 0.999 4）， and Candida tropicalis （R2 = 0.999）. Additionally， the coefficient of variation for the resultant concentration logarithmic values was less than 5%， meeting precision requirements. Furthermore， preliminary validation using clinical specimens demonstrated consistent results compared to clinical culture findings. Conclusion ddPCR exhibits rapidity， high sensitivity， good repeatability， and high specificity in detecting invasive candidiasis in critically ill patients. This study highlights the potential value of droplet digital PCR as a diagnostic tool for invasive candidiasis.

Key words： droplet digital PCR; invasive candidiasis; rapid diagnosis

Cite this article

Zhijie HE , Weichao LI , Minghui HE , Xiaotong CHEN , Zhao LIN , Yaowei. ZHI . Rapid diagnosis of invasive candidiasis by droplet digital PCR[J]. The Journal of Practical Medicine, 2024 , 40(19) : 2738 -2746 . DOI: 10.3969/j.issn.1006-5725.2024.19.013

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