Hietala S K, Crossley B M
California Animal Health and Food Safety Laboratory, University of California, Davis, 95616, USA.
J Clin Microbiol. 2006 Jan;44(1):67-70. doi: 10.1128/JCM.44.1.67-70.2006.
In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37 degrees C and 2 weeks at temperatures ranging from ambient room temperature to -70 degrees C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes.
近年来，针对高致病性病原体的检测职责已从国家参考实验室扩展到地方和区域实验室网络，以支持加强疾病监测并测试应急能力。将特定病原体和高致病性病原体的检测工作扩展到参考实验室之外，这就迫切需要标准化的、无传染性的病原体替代物，用作培训和能力验证测试样本。在本研究中，开发了逆转录-聚合酶链反应（RT-PCR）检测的RNA靶标，并将其包装成铠甲RNA，用作四种高致病性动物病原体的无传染性、可定量的合成替代物，这四种病原体分别是：经典猪瘟病毒；口蹄疫病毒；新泽西血清群水疱性口炎病毒；以及印第安纳血清群水疱性口炎病毒。添加到口腔拭子液体标本中的铠甲RNA在逆转录-PCR检测过程的各个阶段都模拟了病毒阳性临床材料，包括通过四种不同的商业提取程序回收RNA、逆转录、PCR扩增以及在与现有实时PCR检测动态范围一致的靶标浓度下进行实时检测。添加到口腔拭子液体标本中的铠甲RNA浓度在选定的储存条件下保持稳定，这些条件近似于能力验证样本运输和处理预期的时间和温度极端情况，包括在37℃下24小时以及在从室温到-70℃的温度范围内2周。分析测试性能，包括在检测动态范围内的重现性，表明铠甲RNA可为特定检测培训和能力验证测试目的提供无传染性、可定量且稳定的病毒替代物。
