Tuyển tập các báo cáo nghiên cứu khoa học quốc tế về bệnh thú y đề tài: A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples | J. Vet. Sci. 2009 10 1 35-42 DOI JOURNAL OF Veterinary Science A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples 1 2 2 1 Vijayarani Kumanan Sam R. Nugen Antje J. Baeumner Yung-Fu Chang .Animal Health Diagnostic Center Department of Population Medicine and Diagnostic Sciences College of Veterinary Medicine and 2Department of Biological and Environmental Engineering Cornell University Ithaca NY USA A simple membrane-strip-based lateral-flow LF biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction RT-PCR for the detection of a small number ten of viable Mycobacterium M. avium subsp. paratuberculosis MAP cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay RNA was extracted from fecal samples spiked with a known quantity of 101 to 106 MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The dye in the liposomes provides a signal that can be read visually quantified with a hand-held reflectometer or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria such as M. avium complex M. ulcerans M. marium M. kansasii M. abscessus M. asiaticum M. phlei M. fortuitum M. scrofulaceum M. intracellulare M. smegmatis and M. bovis. The .
