Wasswa Fredrickson B, Kassaza Kennedy, Nielsen Kirsten, Bazira Joel
Department of Microbiology and Parasitology, Mbarara University of Science and Technology, Mbarara P.O. Box 1410, Uganda.
Department of Microbiology & Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
J Fungi (Basel). 2025 Jun 4;11(6):430. doi: 10.3390/jof11060430.
is a capsulated yeast that enters the body through inhalation and migrates via the bloodstream to the central nervous system, causing cryptococcal meningitis. Diagnosis methods are culture, serology, and India ink staining, which require cerebrospinal fluid (CSF) or whole blood. Molecular methods are used for epidemiological studies and require expensive commercial DNA extraction kits. This study aimed to develop an economical in-house method for extracting DNA from whole blood. cells of varying McFarland standards were spiked into expired blood, then lysed using laboratory-prepared lysis buffer and ox-bile solution, followed by organic DNA extraction. Ordinary PCR targeting the CNAG 04922 gene was performed. To determine the limit of detection, serial dilutions of were made, and DNA extraction was performed on other parts cultured on yeast extract peptone dextrose agar to determine colony-forming units (CFU). The lysis buffer method successfully extracted DNA from as low as the average of 62 CFU in 0.9 mL of expired blood with superior quality and high yield compared to ox-bile. The lysis buffer method yielded higher DNA quality and quantity than ox-bile and detected low concentrations of in expired blood. This method presents a cost-effective alternative for molecular diagnosis in resource-limited settings.
是一种包膜酵母菌，通过吸入进入人体，并经血流迁移至中枢神经系统，引起隐球菌性脑膜炎。诊断方法包括培养、血清学和墨汁染色，这些方法需要脑脊液（CSF）或全血。分子方法用于流行病学研究，需要昂贵的商业DNA提取试剂盒。本研究旨在开发一种从全血中提取DNA的经济的内部方法。将不同麦氏标准的细胞加入过期血液中，然后使用实验室制备的裂解缓冲液和牛胆汁溶液进行裂解，随后进行有机DNA提取。针对CNAG 04922基因进行普通PCR。为了确定检测限，进行了系列稀释，并对在酵母提取物蛋白胨葡萄糖琼脂上培养的其他部分进行DNA提取以确定菌落形成单位（CFU）。与牛胆汁相比，裂解缓冲液法成功地从低至0.9 mL过期血液中平均62 CFU的样本中提取出高质量和高产量的DNA。裂解缓冲液法产生的DNA质量和数量高于牛胆汁，并能检测过期血液中低浓度的[具体物质未提及]。该方法为资源有限环境下的分子诊断提供了一种经济有效的替代方案。
