Animal Health Department, Neiker-Tecnalia, Berreaga, 1, 48160 Derio, Bizkaia, Spain.
Appl Environ Microbiol. 2011 Dec;77(24):8615-24. doi: 10.1128/AEM.05818-11. Epub 2011 Oct 14.
The growth rate of Mycobacterium avium subsp. paratuberculosis was assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8 M. avium subsp. paratuberculosis strains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.
分枝杆菌属副结核亚种的生长速度通过在补充有 OADC（油酸、白蛋白、葡萄糖、过氧化氢酶）、Tween 80 和分枝菌素 J 的 7H9 培养基中使用不同方法进行评估。通过湿重、浊度测量、活菌计数和定量 PCR（ParaTB-Kuanti；F57 基因）确定 8 株分枝杆菌属副结核亚种（K10、2E、316F、81、445、764、22G 和 OVICAP 49）的世代时间和最大比生长速率。在生长曲线和计算参数方面观察到菌株间的差异。定量方法在特定时间点对每种菌株的结果不同。世代时间从活菌计数和 qPCR 的平均 1.4 天到湿重和浊度测量的大约 10 天不等。湿重、浊度和 ParaTB-Kuanti qPCR 方法彼此之间相关性最好。通常，将活菌计数作为参考方法评估活菌；然而，由于分枝杆菌属副结核亚种的聚集问题和非可培养的分枝杆菌属副结核亚种细胞的存在，我们得出结论，单拷贝基因的 qPCR 可能可用于可靠地快速估计样品中分枝杆菌属副结核亚种的细菌数量。
