Development and evaluation of method to detect rabies virus using nucleic acid sequence based amplification with internal control RNA
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Standard nucleic acid sequence based amplification (NASBA) method without internal control RNA (IC RNA), although uncertain, it may not avoid false negative. The objective of this study as to develop and to evaluate whether NASBA with internal control RNA (IC-NASBA) could be used for diagnosis of rabies and to avoid false negative without compromising its detection sensitivity when compare to NASBA. In this study, the modified overlap extension by polymerase chain reaction method was used to construct rabies modified fragment containing 25 changed and 42 inserted nucleotides in N gene fragment of rabies virus as internal control in IC-NASBA. Segment of changed nucleotide was a target for specific IC probe. Its longer sequences reduced competitive capacity with WT in amplification reaction. Constructed IC RNA was added to each reaction tube in amplification step. It was amplified by the same target NASBA primers and was detected by a probe complementary to the internal nontarget sequences. IC RNA of 25x10[superscript-4] fg per reaction was used in the amplification step. IC-NASBA was applied in the target of brain and urine specimens. Twenty samples of rabies infected and non-infected brains were examined. The result showed that IC RNA could be detected in all except in high quantity virus samples. Fifteen urine samples of them, previously presence to contain inhibitor, were subject to IC-NASBA assay. Results were in accessed with nested RT-PCR. In conclusion, IC-NASBA should be useful in the diagnosis of rabies and may increase accuracy in rabies detection.
无内参RNA（internal control RNA, IC RNA）的基于核酸序列扩增技术（nucleic acid sequence based amplification, NASBA）方法，即便存在不确定性，仍可能无法规避假阴性结果。本研究旨在开发并评估带内参RNA的NASBA（IC-NASBA）是否可用于狂犬病诊断，且在与传统NASBA对比时，能够在不降低检测灵敏度的前提下避免假阴性。本研究采用改良的聚合酶链反应（polymerase chain reaction, PCR）重叠延伸法，构建了狂犬病病毒N基因片段的改造片段作为IC-NASBA的内参：该改造片段在狂犬病病毒N基因片段中引入了25个突变核苷酸与42个插入核苷酸，其中突变核苷酸区段可作为特异性内参探针的靶标，其更长的序列可降低与野生型（wild type, WT）病毒在扩增反应中的竞争能力。将构建完成的IC RNA添加至扩增步骤的每一个反应管中，其可使用与靶标NASBA相同的引物进行扩增，并通过与内参非靶序列互补的探针完成检测。本实验中每反应体系使用的IC RNA用量为25×10^-4 fg。IC-NASBA被应用于脑组织与尿液标本的检测，共检测20份狂犬病感染与非感染的脑组织样本，结果显示除高病毒载量样本外，其余样本均可检测到IC RNA。其中15份此前被检出存在扩增抑制剂的尿液样本接受了IC-NASBA检测，检测结果以巢式RT-PCR（nested RT-PCR）作为参照进行评估。综上，IC-NASBA可有效应用于狂犬病的诊断，且可在不牺牲检测灵敏度的前提下提升狂犬病检测的准确性。
