XÂY DỰNG QUY TRÌNH PHÁT HIỆN NẤM CANDIDA SPP. BẰNG PHƯƠNG PHÁP MULTIPLEX PCR

Abstract

Background: According to the Centers for Disease Control and Prevention (CDC) and the US National Health Care Safety Network, Candida spp. ranked 5th among nosocomial pathogens and 4th among blood-borne pathogens. Currently, this shifting trend in Vietnam is also occurring with the highest infection rates in 04 species of Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. However, the traditional methods of detecting Candida species, although easy to implement, have many disadvantages: dependent on objective factors, and are time-consuming, leading to not quick and timely treatment indications. The Molecular logy diagnostic methods have many advantages in detecting and identifying pathogens. Objectives: This study was carried out with two objectives: (1) Determine and optimize multiplex PCR conditions to detect 04 fungi C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis. (2) Develop a process to simultaneously detect 04 species of C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis by multiplex PCR method. Method: Candida spp. was admitted to the University of Medicine and Pharmacy Hospital in Ho Chi Minh City. Ho Chi Minh City, Le Van Thinh Hospital, and Military Hospital 175 from October 2020 to May 2021. The fungus was detected by the following methods: (1) germplasm test, (2) isolation on CHROMagar Candida medium, (3) optimization of the Candida spp detection procedure by Multiplex PCR technique and using the optimal procedure in detecting Candida spp. from patient samples and (4) sequenced 5 amplification products to check for specificity. Then, compare and interpret the results of detecting Candida spp. by 3 methods: germ tube test, CHROMagar Candida, and Multiplex PCR. Results: 40 patient samples were detected by 3 methods: Germ tube testing: detecting 13 species of C. albicans and 27 species of C. non-albicans, culture of CHROMagar Candida: detecting 18 species of C. albicans, 5 species C. tropicalis, 3 species of C. glabrata and 14 species of C. non-albicans. With multiplex PCR: detected 18 species of C. albicans, 7 species of C. tropicalis, 8 species of C. glabrata, 6 species of C. parapsilosis and 1 unidentified species. Multiplex PCR components for optimal detection of 4 Candida species in 1 effendop tube: PCR buffer 10 X 2.2 µl; MgSO4 25 mM 0.6 µl; Taq DNA polymerase 5 UI/µl 0.3 µl; dNTP 10 mM 0.5 µl; primer F_alb 5 pmol 0.3 µl; R_alb 5 pmol 0.5 µl; F_tro 5 pmol 0.3 µl; F_para 5 pmol 0.6 µl; R_para 5 pmol 0.6 µl; F_gla 5 pmol 0.3 µl, R_gla 5 pmol 0.3 µl; DNA extract 1 µl; enough demineralized water 25 µl. Optimized PCR program: initial denaturation period 95^oC 5 min (1 cycle), stage 2 (40 cycles): denaturation 95^oC 30 s, primer 59 ^oC 30 s, primer extension 72^oC 30 seconds; final elongation n period 72°C 8 min (1 cycle).