Advances in Animal and Veterinary Sciences

Research Article
Adv. Anim. Vet. Sci. 3 (11): 577 - 587
http://dx.doi.org/10.14737/journal.aavs/2015/3.11.577.587
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Falguni Mukherjee1a, Kommoju Nagmani2a, Kota Sri Naga Leela Surendra1, Bhaskaran Mohana Subramanian3, Vijay Sriram Bahekar1, Amitesh Prasad1, Samir Kumar Rana1, Ponnanna Nadikerianda Muthappa1, Girish Kumar Sharma4, Villuppananoor Alwar Srinivasan5*

1National Dairy Development Board, R&D Laboratory, Hyderabad, 500032, Telangana, India; 2Department of Biotechnology, Jawaharlal Nehru Technological University Hyderabad and Research and Development, Indian Immunologicals Limited, Hyderabad, 500032, Telangana, India; 3Translational Research Platform for Veterinary Biologicals, Tamil Nadu Veterinary and Animal Sciences University, Chennai, 600051, India; 4National Dairy Development Board, Anand, 388001, Gujarat, India; 5National Dairy Development Board, Animal Health, Gachibowli, Hyderabad 500032, Telangana, India.

*Correspondence | Villuppananoor Alwar Srinivasan, National Dairy Development Board, Telecom Nagar, Gachibowli, Hyderabad, India; Email: srinivasanva1948@gmail.com

aFalguni Mukherjee and Kommoju Nagmani contributed equally to this work.

Abstract
A diagnostic real-time PCR (qPCR) targeting the Brucella cell salt extractable outer membrane protein gene bcsp-31 was optimized for identification of genus Brucella. The assay had an analytical sensitivity of 30fg and reliably detected up to one copy number of the positive control plasmid construct, and 1x104 Brucella cells/reaction from spiked bovine tissue matrices. The qPCR detected DNA from 30 Brucella strains but not from non-Brucella strains. The qPCR was reliable, reproducible and could be completed in 72 minutes. Comparative quantification of Brucella copy number was established by utilizing normalized Cq values. The best return of validation estimates were obtained when animal-wise results of qPCR were compared to the combined status of culture and serology (n=230) since the two assays were strongly associated (κ =0.848 at 95% CI) and revealed a diagnostic sensitivity (DSe) of 77.8% and specificity (DSp) of 100%, positive and negative predictive (PPv and NPv) value of 100% and 94.61% at 95% CI, respectively. In contrasts, the DSe, DSp, PPv and NPv values obtained after comparison of results of qPCR and culture were 100%, 86.55%, 18.2% and 100% at 95% CI, respectively. Therefore, if the estimates were assessed in parallel, together they could form a reliable and rapid diagnostic tool for screening bovine brucellosis.

Keywords | Brucellosis, bcsp-31, qPCR, conventional PCR

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | August 01, 2015; Revised | August 20, 2015; Accepted | August 20, 2015; Published | October 05, 2015

Citation | Mukherjee F, Nagmani K, Surendra KSNL, Subramanian BM, Bahekar VS, Prasad A, Rana SK, Muthappa PN, Sharma GK, Srinivasan VA (2015). Optimization and validation of a diagnostic real-time PCR for bovine brucellosis. Adv. Anim. Vet. Sci. 3(11): 577-587.

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2015 Mukherjee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.