Partitioning and probe-based quantitative PCR assays for the wastewater monitoring of <i>Mycobacterium tuberculosis</i> complex, <i>M. tuberculosis</i> , and <i>M. bovis</i>

Three new probe-based quantitative PCR assays were designed based on existing PCR assays to quantitate <i>Mycobacterium tuberculosis</i> complex (MTBC) species, <i>M. tuberculosis</i> (MTB), and <i>M. bovis</i> (MB) in wastewater targeting genomic regions <i>rv0577</i>, RD9, and the deletion of RD4, respectively. The assays were validated for specificity using four Mycobacterial species, including two MTBC species and two non-tuberculosis Mycobacteria species, and endogenous wastewater samples from Ottawa, Ontario, Canada, Mumbai, India, and a remote Northern Indigenous community in Nunangat with known ongoing tuberculosis cases or outbreaks. The three assays demonstrate high sensitivity and are suitable for use in wastewater. Partitioning experiments performed on endogenous MTBC and MTB in collected wastewaters from Mumbai, India with known tuberculosis outbreaks show that the targeted genomic regions of <i>rv0577</i> (MTBC) and RD9 (MTB) used to quantitate human tuberculosis infection predominately partition to solids fraction of wastewaters. The partitioning results of this study, in combination with the presented probe-based PCR assays, provide guidance on how to best enrich wastewaters and rapidly and economically quantify tuberculosis with high specificity and sensitivity in wastewaters.