一步法RT-PCR和两步法RT-PCR对蜜蜂病毒诊断研究的比较
Research on the detection of bee viruses by one step and two step RT-PCR
贾慧茹1** 周 婷1 高夫超2***
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DOI：10.7679/j.issn.2095-1353.2015.038
作者单位：1. 中国农业科学院蜜蜂研究所，农业部授粉昆虫生物学重点开放实验室，北京100093；2. 黑龙江省农业科学院牡丹江分院，牡丹江 157041
中文关键词：蜜蜂病毒，一步法RT-PCR，两步法RT-PCR
英文关键词： bee virus, one-step RT-PCR, two-step RT-PCR
中文摘要：
【目的】 探讨适合蜜蜂病毒病诊断的RT-PCR技术。【方法】 从感染蜜蜂以色列急性麻痹病毒、残翅病毒、囊状幼虫病毒、急性麻痹病毒、黑蜂王台病毒以及慢性麻痹病毒的阳性样品中，分别提取这6种病毒的RNA。然后，同时应用一步法RT-PCR和两步法RT-PCR的反应体系对6种病毒扩增，并对两种方法的灵敏性进行比较和分析。【结果】 上述两种方法分别得到158 (IAPV)、269 (DWV)、342 (SBV)、460 (ABPV)、536 (BQCV)和774 bp(CBPV)的扩增片段，测序结果证实扩增片段符合预期。两步法RT-PCR可检测到更低浓度的病毒颗粒。【结论】 结果表明，该2种方法均可快速、有效地诊断蜜蜂病毒。但两步法RT-PCR灵敏性更高。
英文摘要：
[Objectives] To find which RT-PCR technology can effectively detect bee viruses. [Methods] Bee viruses (IAPV, DWV, SBV, ABPV, BQCV, and CBPV) were extracted from bees and the sensitivity of one and two step RT-PCR in detecting these six virus was compared. [Results] Both methods amplified viral fragments, which were 158 (IAPV), 269 (DWV), 342 (SBV), 460 (ABPV), 536 (BQCV) and 774 bp (CBPV), in length. Serial dilutions assay showed that two-step RT-PCR detected much lower concentrations of virus particles. [Conclusion] Comparison of one-step and two-step RT-PCR indicates that both methods can effectively detect many bee viruses under optimized conditions, however, two-step RT-PCR is more sensitive than one-step RT-PCR.