title: Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory.
abstract: This study aimed to develop a method for rapid and accurate identification of yeasts obtained in the clinic, especially from immunocompromised patients, in order to provide a timely and appropriate antifungal therapy. A total of 112 Candida isolates were analyzed in this study; 28 of them were used to validate the PCR-HRM method in species identification in a blinded manner. Strains were identified by conventional techniques that use VITEK 2 YST cards and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). These methods were compared to the newly developed technique based on real-time polymerase-chain reaction high-resolution melting (PCR-HRM). Discordant results were resolved with internal transcribed spacer (ITS) gene sequencing, the "golden standard" used to evaluate the reliability of all methods in identifying yeasts at the species level. PCR-HRM sensitivity was assessed with the isolated strains. VITEK 2, MALDI-TOF-MS, and PCR-HRM accurately identified 89.2% (74/83), 97.6% (81/83), 100% (83/83) of the isolates, respectively. PCR-HRM detection limit was 1fg/μl of yeasts. In validation assays, a 100% accuracy rate was achieved by the use of PCR-HRM. Therefore, the PCR-HRM method is a rapid, sensitive, and specific diagnostic approach, which provides a cost-effective and more suitable alternative for yeast identification in a clinical laboratory. Future research is needed for automation of data acquisition.
authors.affiliation: Postgraduate Training Base of General Hospital of Shenyang Military Command, Liaoning Medical University, Jinzhou, China.
authors.forename: Guo
authors.initials: G
authors.lastname: Ninghui
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China.
authors.forename: Wang
authors.initials: W
authors.lastname: Bing
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China.
authors.forename: Ren
authors.initials: R
authors.lastname: Wei
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Mengmeng
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China.
authors.forename: Chu
authors.initials: C
authors.lastname: Meiling
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Dongya
authors.affiliation: The Clinical Laboratory, The First Hospital of Lanzhou University, Gansu, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Liqiong
authors.affiliation: The General Hospital of Shenyang Military, Shenyang, China 13309884078@189.cn ylq8482@163.com.
authors.forename: Xue
authors.initials: X
authors.lastname: Wencheng
mesh_terms: D002175:Candida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D009172:Mycology / Q000379:methods*; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D015003:Yeasts / Q000302:isolation & purification
keywords: Candida; Polymerase-chain reaction high-resolution melting analysis (PCR-HRM); Rapid identification