Mycobacterium avium subsp. paratuberculosis as a template in the evaluation of automated kits for DNA extraction from bovine organs

Abstract

Molecular diagnostic tests are widely implemented in animal and human health microbiology. Efficient nucleic acid extraction methods are essential in diagnostic laboratories and automation is a valuable tool for those with high throughput activity. Nucleic acid extraction protocols present variable efficiency depending on the composition of the specimen and the chemical-physical characteristics of the target pathogen. In the present study, we compared the DNA extraction performances of four automated methods (kits I, M, P, Q) adapted on a Hamilton Robotics ''Microlab Starlet'' extraction unit, and one manual method (kit R). Ten-fold dilutions of Mycobacterium avium subsp. paratuberculosis (MAP) were used to contaminate bovine central nervous system (CNS) and lung-spleen-liver pools (LSL). In consideration of its chemical-physical characteristics, MAP was selected as a template for pathogen DNA detection. Analytical performance and repeatability were assessed through downstream real-time PCR amplification, hands-on time (HOT), total turnaround time (TAT) and costs of all kits. MAP was detected differently depending on extraction kit and type of matrix analysed. Kits M and I showed the highest analytical performance on CNS (1 MAP/ml) and LSL (10 MAP/ml), respectively. Besides analytical results, kits I and M displayed high repeatability, the same HOT, very similar TAT, and were inexpensive. In conclusions, different standardized automated systems have been established with high throughput, sensitivity and repeatability for CNS and LSL. Our results also demonstrated that is necessary to assess the effectiveness of extraction kits in matrices not previously tested to avoid the risk of unreliable diagnostic outcomes.

Serum Metabolomic Profiles of Paratuberculosis Infected and Infectious Dairy Cattle by Ambient Mass Spectrometry

“…The same PCR protocol was used for both confirmatory test and direct analysis. In brief, 300 μl of supernatant or culture medium was subjected to a bead-beating step in order to enhance MAP DNA recovery as previously described ( 36 ). DNA extraction was performed manually with the High Pure PCR preparation kit (Roche Diagnostic, Mannheim, Germany) or by the automated MagMAX™ 96 Viral Isolation Kit (Ambion, Austin, USA) using the Microlab Starlet automated extraction platform (Hamilton Robotics, Bonaduz, Switzerland) or the KingFisher Flex instrument (ThermoFisher Scientific Inc., Worcester, MA, USA).…”

Metabolic signature of Mycobacterium avium subsp. paratuberculosis infected and infectious dairy cattle by integrating nuclear magnetic resonance analysis and blood indices

“…Individual fecal samples were collected from the rectal ampulla and analyzed applying microbiological and molecular diagnostic methods for MAP identification. All samples were processed for testing by IS900 direct real-time PCR (qPCR, Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) according to Pozzato et al ( 28 , 29 ) while the culture was carried out by a double decontamination method on modified Middlebrook 7H9 liquid media (7H9+). After 6 weeks of culture, 7H9+ broths were examined by Ziehl-Nielsen staining and real-time PCR, as reported by our previous work (27a).…”