Tan Jianming, Cooke Julie, Clarke Nadeene, Tannock Gregory A
Department of Biotechnology and Environmental Biology, School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia.
J Virol Methods. 2008 Feb;147(2):312-8. doi: 10.1016/j.jviromet.2007.09.011. Epub 2007 Oct 31.
A real-time PCR was used to measure increases in viral DNA in Marek's disease virus (MDV)-infected primary chicken cell cultures in order to optimize methods for viral isolation. Serotype-1 and -3 vaccine and serotype-1 challenge strains exhibited similar growth characteristics, with increases in viral DNA being proportional to inoculum size. Studies of viral growth revealed a linear relationship between increase in MDV copy number and infectious titre, although the rate of increase for copy number was greater. Using real-time PCR, viral DNA yields of the virulent Woodlands strain in infected chicken kidney cultures were shown to be slightly, but not significantly, higher than in chicken embryo kidney cultures and significantly higher than in chicken embryo fibroblast cultures. Viral DNA levels in freshly trypsinised cells suspended in growth medium and infected with the Woodlands strain were higher than levels obtained following the inoculation of monolayer cultures. For cells infected in suspension, no significant enhancement of yield was observed following a medium change after 2-3 days. Peak yields were obtained at days 6-8 after inoculation of all cultures. Findings obtained from the optimization of viral DNA levels were applied to a program for the isolation of Australian strains of serotype-1 viruses from problem flocks over 3 years. Significant improvements were obtained in the isolation rate of strains capable of growing to high titre (>10(4) plaque-forming units/mL) for use in challenge studies.
采用实时荧光定量聚合酶链反应（real-time PCR）来检测感染马立克氏病病毒（MDV）的原代鸡细胞培养物中病毒DNA的增加情况，以优化病毒分离方法。1型和3型疫苗株以及1型强毒株表现出相似的生长特性，病毒DNA的增加与接种量成正比。病毒生长研究表明，MDV拷贝数的增加与感染滴度之间存在线性关系，尽管拷贝数的增加速率更大。通过实时荧光定量聚合酶链反应发现，在感染的鸡肾培养物中，强毒林地株的病毒DNA产量略高于鸡胚肾培养物，但差异不显著，且显著高于鸡胚成纤维细胞培养物。悬浮于生长培养基中并感染林地株的新鲜胰蛋白酶消化细胞中的病毒DNA水平高于单层培养物接种后的水平。对于悬浮感染的细胞，在2 - 3天后更换培养基后，未观察到产量有显著提高。所有培养物在接种后第6 - 8天获得最高产量。从优化病毒DNA水平获得的结果应用于一个为期3年的从问题鸡群中分离澳大利亚1型病毒株的项目。在能够生长到高滴度（>10⁴ 蚀斑形成单位/毫升）用于攻毒研究的毒株分离率方面取得了显著提高。
