Bogomolni Andrea L, Frasca Salvatore, Matassa Keith A, Nielsen Ole, Rogers Kara, De Guise Sylvain
1 Department of Pathobiology and Veterinary Science, University of Connecticut, 61 N Eagleville Rd., Storrs, Connecticut 06269, USA.
J Wildl Dis. 2015 Apr;51(2):454-65. doi: 10.7589/2014-05-142. Epub 2015 Feb 3.
Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.
在全球范围内，搁浅的海洋哺乳动物以及应对海洋哺乳动物死亡事件的网络人员提供了许多有关海洋麻疹病毒感染的信息。一种用于确定组织样本中病毒含量的检测方法，将有助于动物健康的常规监测以及大规模死亡事件的监测。质量不佳的样本导致的假阴性结果妨碍了对感染真实程度的判定，而在样本采集时可能仅有少量组织样本或存档RNA可供未来进行回顾性分析。我们基于Taqman探针技术开发了一种一步法双重实时逆转录定量PCR检测方法（RT-qPCR），用于定量2006年在美国东北海岸港湾海豹（Phoca vitulina concolor）和灰海豹（Halichoerus grypus）疫情中分离出的海豹瘟热病毒（PDV）。选择甘油醛-3-磷酸脱氢酶（GAPDH）基因来评估RNA质量。这种双重检测方法对PDV具有特异性，在10(0)至10(9)拷贝的双链质粒DNA范围内具有敏感性。对于GAPDH靶标，双重反应可扩增1至10(9)拷贝的双链质粒DNA，并且在多种海豹物种中均可检测到。该检测方法降低了因组织降解和孔间差异导致假阴性结果的可能性，同时提供了对PDV的灵敏且特异的检测，适用于涉及多种海豹物种的分子流行病学研究以及现场和实验室调查中的病原体检测。
