Direct PCR on tissue samples to detect Mycobacterium tuberculosis complex: an alternative to the bacteriological culture
Investigation article published in Journal of Clinical Microbiology
January 21st, 2021
Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union (EU). 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, comparatively 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 inter-laboratory trial between EU National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic. Diagnostic sensitivity and specificity was, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as M. marinum and "M. avium subsp. hominissuis" species, and Whole Genome Sequence 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 programmes
Lorente-Leal V., Liandris E., Pacciarini M., Botelho A., Kenny K., Loyo B., Fernandez R., Bezos J., Dominguez L., de Juan L. and Romero B..
 | Servicio de Micobacterias (MYC). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM). |
| Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM). |
| Centro Nazionale di Referenza per la Tubercolosi Bovina. Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia Romagna (IZSLER). | |
| Instituto Nacional de Investigação Agrária e Veterinária (INIAV). | |
| Central Veterinary Research Laboratory (CVRL). | |
| Department of Agriculture, Food and the Marine (DAFM). | |
 | Servicio de Sanidad Animal. Junta de Castilla y León. |
| Consejería de Agricultura y Ganadería. Junta de Castilla y León. |
 | Ministerio de Agricultura, Pesca y Alimentación (MAPA). |
