Mycoplasma detection by triplex real-time PCR in bronchoalveolar lavage fluid from bovine respiratory disease complex cases

BMC Veterinary Research

Research Article

Engbert A. Kooi¹  Miriam G. J. Koene¹  Fimme J. van der Wal¹  Freddy M. de Bree¹  Alex Bossers¹  Bregtje Smid¹  Adriaan F. Antonis¹  Henk J. Wisselink¹  Jan B. W. J. Cornelissen¹ 

[1] Department of Infection Biology, Central Veterinary Institute of Wageningen UR, P.O. Box 65, 8200, Lelystad, AB, The Netherlands;

关键词: Mycoplasma;    M. dispar;    M. bovis;    M. bovirhinis;    Triplex PCR;    RespoCheck;    Bovine respiratory disease;   

DOI  :  10.1186/s12917-017-1023-6

 received in 2016-09-29, accepted in 2017-04-03,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundIn this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar, M. bovis and M. bovirhinis, all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species.ResultsThe analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar, and 30 cfu/mL for M. bovis or M. bovirhinis. The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo rM. bovis and M. bovirhinis. The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was, as determined by testing Mycoplasmas strains (n = 17) and other bacterial strains (n = 107), 100, 98.2 and 99.1% for M. bovis, M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis, M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves.ConclusionIn conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.

【 授权许可】

CC BY   
© The Author(s). 2017