Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium Tuberculosis by Droplet Digital PCR

作者：Yang Zheng, Hui Xia, Xundi Bao, Bing Zhao, Ping He, Yanlin Zhao · 发表于：Infection and Drug Resistance · 年份：2022 · DOI：10.2147/idr.s381097 · 被引用次数：16 · 研究领域：Tuberculosis Research and Epidemiology、Mycobacterium research and diagnosis、Innovative Microfluidic and Catalytic Techniques Innovation

Purpose: The drug resistance of Mycobacterium tuberculosis constitutes a major public health threat. Existing approaches make it challenging to detect low levels of drug-resistant TB, also known as heteroresistance (HR), in a population. The recently found droplet digital PCR (ddPCR) is a sensitive method for determining the precise amount of nucleic acid in a sample. We used ddPCR to test the Mycobacterium tuberculosis heteroresistance because it delivers more exact quantitative data without the need for a reference curve. Patients and Methods: A TaqMan-MGB probe mutation detection assay was developed in order to determine the mutant and wild-type sequences of the isoniazid resistance katG (315) gene. We produced heteroresistant MTB combinations, which were subsequently identified by ddPCR, qPCR, and MeltPro/INH. In addition, 21 clinical sputum samples with positive smears were used to validate each method’s capacity to determine HR in sputum. Results: We discovered that ddPCR can detect mutant sequences in as few as 0.01% of a combination. DeepMelt TB/INH, which is less sensitive in comparison, cannot detect HR with high resolution and requires a mutation rate of 50% to identify. qPCR likewise has a high resolution of 0.02%, but unlike ddPCR, it cannot determine the exact number of mutations. Our assay is applicable to sputum as well. ddPCR found a katG 315 substitution in two sputums with extremely low values of HR (0.26% and 0.14%). In 21 samples of clinical sputum, the H...