白念珠菌唑类耐药分子检测体系的建立  

Establishment of a Molecular Detection System for Azole Resistance in Candida albicans

作　　者：王梦凡[1,2] 王旭 刘春龙 齐崴[1] Wang Mengfan;Wang Xu;Liu Chunlong;Qi Wei(School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350,China;School of Life Sciences,Tianjin University,Tianjin 300072,China;Dynamiker Biotechnology(Tianjin)Co.,Ltd.,Tianjin 300457,China)

机构地区：[1]天津大学化工学院,天津300350 [2]天津大学生命科学学院,天津300072 [3]丹娜(天津)生物科技股份有限公司,天津300457

出　　处：《天津大学学报(自然科学与工程技术版)》2025年第4期414-422,共9页Journal of Tianjin University:Science and Technology

基　　金：国家自然科学基金资助项目(22178260);天津市科学技术局重大专项资助项目(20ZXGBSY00040).

摘　　要：随着免疫抑制剂与侵入性手术的应用,侵袭性真菌病的发病率逐渐上升,而由此出现的抗真菌药物大量使用也导致了真菌耐药性的增强.其中,白念珠菌(Candida albicans)对唑类药物耐药性日益明显,这对开发早期临床耐药性诊断方法提出了更大的需求.相关研究显示,白念珠菌的耐药性涉及到多种机制,其中erg11基因上的热点区突变是其产生唑类耐药性的主要机制之一.本研究旨在基于多色探针熔解曲线分析技术(MMCA)建立一种针对白念珠菌唑类耐药的分子检测方法,从而对3个热点突变区内的4种主要的突变类型进行基因分型.研究纳入了11株耐药和22株敏感的白念珠菌,通过对其进行药敏检测、耐药相关基因突变类型鉴定,建立真菌耐药表型与耐药基因型之间的联系.药敏结果显示,11株耐药株和22株敏感株的最低抑菌浓度(MIC)值分别为16~64μg/mL和0.5~1.0μg/mL.耐药基因型鉴定结果显示有erg11HS1热点区突变的菌株9株,erg11HS3热点区突变的菌株2株,未能鉴定到erg11HS2热点区突变的菌株,相关突变型与耐药性之间的联系符合预期结果.在此基础上,通过设计数对特异性引物和荧光探针,并筛选最佳反应程序,建立了一种鉴定有关突变型与野生型的MMCA检测体系.对该检测体系的效果进行了评估和验证:所有4种突变型和野生型均被准确分型,两者的溶解温度差异均大于3.5℃,检出限最低可达1.9×10^(2)拷贝数/mL,扩增效率均大于90%.在33例白念珠菌的样本中,以一代测序为标准,该体系检测的一致率为93.94%(31/33),不同物种间无特异性交叉.本研究为白念珠菌病的早期唑类药物耐药性诊断提供了新方法.With the increasing use of immunosuppressive agents and invasive surgeries,the incidence of invasive fungal diseases is gradually increasing,leading to an increased prevalence of antifungal drug resistance due to extensive use of these drugs.Candida albicans,in particular,has shown a gradual increase in resistance to azole antifungal drugs,which highlights the need for early detection of drug resistance.Studies have indicated that resistance mechanisms in C.albicans involve multiple factors,with hotspot mutations in erg11 being one of the main contributors to azole resistance.This study aimed to establish a molecular detection system using multicolor melting curve analysis(MMCA)for azole resistance in C.albicans via genotyping four major mutation types within three hotspot regions.A total of 11 strains of drug-resistant and 22 strains of drug-sensitive C.albicans were included.The correlation between fungal drug resistance phenotypes and genotypes was established by performing antimicrobial susceptibility testing and identifying mutations in drug resistance-related genes.Drug susceptibility testing revealed that the minimum inhibitory concentration(MIC)values for the 11 resistant and 22 sensitive strains were in the ranges of 16—64μg/mL and 0.5—1.0μg/mL,respectively.Genotypic resistance analysis identified nine strains with mutations in the erg11HS1 hotspot region,two strains with mutations in the erg11HS3 hotspot region,and no strains with mutations in the erg11HS2 hotspot region.Based on this,a MMCA detection system was established to identify mutations and wild types by designing pairs of specific primers and fluorescent probes,and screening for the optimal reaction conditions.The performance of the MMCA detection system was evaluated and validated.All four mutation types and the wild type were accurately genotyped,and the difference in melting temperature between them was more than 3.5℃.The detection limit was as low as 1.9×10^(2) copies/mL,and the amplification efficiency was consistently more than

关 键 词：白念珠菌 耐药性 探针熔解曲线分析 突变型 

分 类 号：TQ426.97[化学工程]