Validating MTBC Nested PCR Specificity

We validated specificity of the MTBC nested PCR with IS6110-specific primers using granuloma lung tissues from a naturally M. tuberculosis–infected long-tailed macaque (Figure 1). We confirmed M. tuberculosis infection in the macaque by mycobacterium culture and interferon gamma release assay, using methods reported elsewhere (24 (link)). We collected and extracted granuloma lung tissues for genomic DNA using a QIAGEN Virus/Pathogen Mini Kit. We purified the products obtained from 181-bp nested PCR testing of the naturally M. tuberculosis–infected macaque and the 19 nested PCR–positive samples randomly selected from populations of wild rhesus macaques from Ban Phon Kor and Ban Sang School and Burmese long-tailed macaques from Tham Pra Khayang and Mangrove Forest Research Center using the GenUP Exo Sap Kit (Biotechrabbit, https://www.biotechrabbit.com) and submitted the samples to Macrogen (https://www.macrogen.com) for DNA sequencing. We aligned nucleotide sequences with published M. tuberculosis sequences accessed from GenBank using MEGA X software (25 (link)). After all amplicons from the M. tuberculosis sequences from macaques in our study showed 100% homology with published sequences, we used an IS6110-specific nested PCR protocol to determine the sensitivity of the M. tuberculosis nested PCR technique, using genomic DNA of the M. tuberculosis H37Rv (ATCC27294) strain, serially diluted (1:10) from 1 ng to 1 fg (26 (link)). We designated the lowest concentration of M. tuberculosis H37Rv that we could detect by nested PCR as the limit of detection (LOD) for this technique.