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3. Optimization and Validation of a Diagnostic Real-Time PCR for Specific Detection of Mycobacterium avium Subspecies Paratuberculosis

Author

Lakshmi, Bonda Rama, primary, Mukherjee, Falguni, additional, Surendra, Kota Sri Naga Leela, additional, Bahekar, Vijay Shriram, additional, Prasad, Amitesh, additional, Rana, Samir Kumar, additional, Nagmani, Kommoju, additional, Muthappa, Ponnanna Nadikerianda, additional, Sharma, Girish Kumar, additional, and Srinivasan, Villuppananooralwar, additional

Published
2015
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4. Development and laboratory validation of duplex real-time PCR for simultaneous detection of Brucella and bovine alphaherpesvirus from clinical specimens.

Author

Saarangi LN, Polapally S, Rana SK, Bahekar VS, Surendra KSNL, Chandrasekhar Reddy RV, Raichur AS, Muthappa PN, and Sharma GK

Subjects
Animals, Brucella genetics, Brucellosis complications, Brucellosis diagnosis, Cattle, Cattle Diseases blood, Herpesviridae Infections complications, Herpesviridae Infections diagnosis, Herpesvirus 1, Bovine genetics, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity, Brucella isolation & purification, Brucellosis veterinary, Cattle Diseases diagnosis, Herpesviridae Infections veterinary, Herpesvirus 1, Bovine isolation & purification
Abstract

A duplex real‑time PCR was developed and validated for the simultaneous detection of Brucella and bovine alphaherpesvirus‑1 (BoHV‑1) from bovine clinical specimens. The bcsp31 gene of Brucella and gB gene of BoHV‑1 were used as targets in the assay. The limit of detection for BoHV‑1 was 0.03 TCID50 of virus and 10 plasmid copies containing the target gene while for Brucella it was 4.1 × 101 CFUs. Intra‑assay and inter‑assay values showed high repeatability and reproducibility of the assay. The diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. The dsn and dsp for detection of Brucella were found to be 95.24% and 95.65%, respectively whereas for BoHV‑1, the dsn (100%) and dsp (99.47%) were slightly higher. The duplex assay had a very good degree of agreement with the respective individual real‑time PCR test {kappa value 0.97 for Brucella and 0.95 for BoHV‑1}. The results of the current study suggest that the duplex assay would be a cost‑effective and time‑saving alternative for the individual real‑time PCR assay for the detection of Brucella and BoHV‑1.

Published
2020
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