Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows

Abstract

The causative agent of bovine tuberculosis (BTB) is Mycobacterium bovis, a bacterium belonging to the M. tuberculosis complex (MTC). The definitive diagnosis is achieved through isolation and identification of M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the "gold standard". This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in milk of positive skin test cows, using primers that were previously tested and proven reliable to target the IS6110 element. Milk previously seeded with M. bovis was used as the starting material, for standardization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme), phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150) of the milk samples tested. This procedure could be used to assist the in vivo diagnosis of BTB, complementing sorological or microbiological tests, becoming an alternative option for epidemiological studies of BTB transmission and preventing contaminated milk from entering the food supply.Keywords: Bovine tuberculosis. Molecular biology. Mycobacterium tuberculosis complex. Milk. ResumoO agente causador da tuberculose bovina (BTB) é o Mycobacterium bovis, uma bactéria pertencente ao complexo M. tuberculosis (CMT). O diagnóstico definitivo é realizado através do isolamento e identificação de M. bovis em amostras clínicas, utilizando uma combinação de cultura bacteriológica e métodos bioquímicos, que são considerados como "padrão de ouro". Entretanto, esses procedimentos são trabalhosos e demorados. No presente estudo, foi avaliado um ensaio de PCR para a detecção direta de DNA de CMT em leite de vacas positivas ao teste cutâneo, utilizando primers previamente testados e comprovadamente confiáveis, para amplificação da região de interesse IS6110. Leite previamente contaminado com M. bovis foi utilizado como material de partida para a padronização da técnica. O procedimento envolveu a extração do DNA por lise enzimática (proteinase K e lisozima), fenol, clorofórmio, álcool isoamílico, seguido de precipitação com etanol e PCR. O ensaio de PCR detectou BTB em leite artificialmente contaminado, com um limite de detecção de 100 UFC/mL, e também foi capaz de detectar o bacilo em 50% (75/150) das amostras de leite testadas. A técnica de PCR pode ser utilizada para auxiliar o diagnóstico in vivo da BTB, além de complementar os testes sorológicos ou microbiológicos, tornando-se uma alternativa para estudos epidemiológicos da transmissão de BTB, prevenindo que o leite contaminado entre na cadeia de alimentos.Palavras-chave: Tuberculose bovina. Biologia molecular. Complexo Mycobacterium tuberculosis. Leite.

Miniaturized Continuous-Flow Digital PCR for Clinical-Level Serum Sample Based on the 3D Microfluidics and CMOS Imaging Device

“…Considering its rapid development over the past few decades, qPCR technology has been used for the diagnosis of many diseases [2,4,[6][7][8][9]. However, there are many factors that affect the amplification efficiency during PCR amplification.…”

A digital PCR system based on the thermal cycled chip with multi helix winding capillary

“…It can amplify nucleic acid molecules from one to several million in a very short period of time (usually in tens of minutes), so it is regarded as the most important molecular biology technology today. It plays an important role in the fields including next-generation high-throughput sequencing and clinical nucleic acid marker detection [1][2][3][4][5][6][7][8][9][10][11][12][13][14] .…”