Evaluation of DNA Extraction Techniques for Detecting Mycobacterium tuberculosis Complex Organisms in Asian Elephant Trunk Wash Samples

Abstract

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

Tuberculosis surveillance of elephants (Elephas maximus) in Nepal at the captive-wild interface

“…The use of DNA methodologies, such as polymerase chain reaction (PCR), may have promising applications along with novel technologies such as detection of volatile organic compounds associated with mycobacterial infection (Kay et al 2010;Nol et al 2014;Crawshaw et al 2014).…”

Direct DNA Modified CTAB Preparation from Nasal Exudate in Live M. bovis Infected Cattle in Mexico Provide with a Valuable Assay Extrapolated to Humans TB Diagnostic Test

“…Detection, identification and differentiation of members of the MTBC complex rely in specificity, sensivity and accuracy of the methods that have been developed since the decades of the 90's. Despite this, still in endemic areas developing countries tuberculin field test as well as conventional techniques (histopathology and bacteriology) are performed due primarily to the costs and availability [25][26][27][28][29][30][31]. Therefore, it is a urgent need for endemic regions to have a routine assay to boost field test (false positive and negative tests) in live cows [37].…”

“…Therefore, it is a urgent need for endemic regions to have a routine assay to boost field test (false positive and negative tests) in live cows [37]. Moreover, the development of an optimal PCR methods that can boost field tuberculin tests (false positive/false negative) of live cows while avoiding unnecessary sacrifice of animals a molecular method should fulfill some basic requirements such as the high quality DNA, the regions to be amplified and primers design [8,16,22,[24][25][26][27][28][29][30][31][37][38][39]. M. bovis detection either from cattle or humans samples have moderate sensitivity, mainly attributed to the difficult for DNA extraction (bacterial lysis, long manipulation steps) or quality of the sample collection [17,[25][26][27][28].…”

“…Moreover, the development of an optimal PCR methods that can boost field tuberculin tests (false positive/false negative) of live cows while avoiding unnecessary sacrifice of animals a molecular method should fulfill some basic requirements such as the high quality DNA, the regions to be amplified and primers design [8,16,22,[24][25][26][27][28][29][30][31][37][38][39]. M. bovis detection either from cattle or humans samples have moderate sensitivity, mainly attributed to the difficult for DNA extraction (bacterial lysis, long manipulation steps) or quality of the sample collection [17,[25][26][27][28]. Nowadays, there are several molecular methods for the detection of M. bovis that goes from simple PCR to multiplex real time PCR using either milk, semen, urine or nasal exudate [16,23,[24][25][26][27][28][29][30][31], some of them fill one or two of the basic requirements mentioned above, but not all.…”

“…The authors concluded that the PCR technique has a better analytical performance than the traditional culture for the detection of mycobacteria in nasal exudates of bovines [24]. To note is the several advancements for the improvement of detection, differentiation of the members of the MTB complex [25][26][27][28][29][30][31], each other have provided with a key point to take in the account either for TBb or human tuberculosis [25][26][27][28][29][30][31].…”