It is important to accurately quantify Mycobacterium tuberculosis (Mtb) load in laboratory-based tuberculosis (TB) research. This study aims to determine if real-time quantitative PCR (qPCR) and digital PCR (dPCR) can be used instead of colony forming unit (CFU) enumeration, to quantify Mtb load in rhesus and cynomolgus macaque tissue samples. Tissue samples of actively infected high Mtb-burden rhesus and cynomolgus macaques were selected from historic sample collections. CFUs were enumerated by plating, and Chelex-extracted genomic DNA used to quantify bacterial load by qPCR and dPCR. Three genes, sigA, 16S and CFP10, were assessed for their ability to quantify Mtb. All genes showed comparable quantification of Mtb between 2 and 20 000 copies/μl in the qPCR and 5–4000 copies/μl in the dPCR assay. The highest bacterial load was observed with dPCR, followed by qPCR, and CFU enumeration. Although the CFU count was consistently lower than the genomic copy numbers predicted by qPCR and dPCR, a significant correlation was observed. Quantification of Mtb by PCR was, however, only possible in higher-Mtb-load samples, suggesting that qPCR and dPCR quantification assays can predict bacterial load in actively infected and higher-Mtb-burden macaque tissue samples.

在基于实验室的结核病 (TB) 研究中准确量化结核分枝杆菌( Mtb ) 载量非常重要。本研究旨在确定是否可以使用实时定量 PCR (qPCR) 和数字 PCR (dPCR) 代替菌落形成单位 (CFU) 计数，以量化恒河猴和食蟹猴组织样本中的Mtb负荷。活跃感染的高Mtb负荷恒河猴和食蟹猴的组织样本选自历史样本集合。通过电镀对 CFU 进行计数，并使用 Chelex 提取的基因组 DNA 通过 qPCR 和 dPCR 量化细菌载量。三个基因，sigA、16S和CFP10，评估了他们量化Mtb的能力。所有基因在 qPCR 中显示出 2 到 20 000 拷贝/μl 之间的Mtb定量，在 dPCR 测定中显示 5-4000 拷贝/μl。dPCR 观察到的细菌载量最高，其次是 qPCR 和 CFU 计数。尽管 CFU 计数始终低于 qPCR 和 dPCR 预测的基因组拷贝数，但观察到显着的相关性。然而，通过 PCR对Mtb进行定量只能在较高Mtb负荷的样本中进行，这表明 qPCR 和 dPCR 定量分析可以预测活跃感染和较高Mtb负荷的猕猴组织样本中的细菌负荷。