Direct PCR on Tissue Samples To Detect Mycobacterium tuberculosis Complex: an Alternative to the Bacteriological Culture

Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union. Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparative to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an interlaboratory trial between European Union National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic equivalents. Diagnostic sensitivity (Se) and specificity (Sp) were, respectively, 96.45% and 93.66%, and the overall agreement (kappa) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as Mycobacterium marinum and Mycobacterium avium subsp. hominissuis, and whole-genome sequencing (WGS) analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture, and the strategy of use should be defined depending on the control or eradication programs.

Automated summary

This study evaluated the analytical and diagnostic performance of a real-time PCR technique targeting bovine tuberculosis, showing higher sensitivity and faster detection compared to traditional culture methods. Despite some cross-reactivity issues, this method is considered a valuable alternative to culture.