PDF(Pubmed)
Abstract：
Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.
摘要：
验证分子检测方法的包容性可以指示病毒感染诊断的可靠性，因为病毒病原体倾向于进行序列变异。这项研究旨在选择基于逆转录定量PCR（RT-qPCR）测定的包容性探针，以诊断感染蜜蜂的最广泛和有害的病毒。即急性蜜蜂麻痹病毒（ABPV），黑色女王细胞病毒（BQCV），慢性麻痹蜂病毒（CBPV），畸形机翼病毒变体A（DWVA）和B（DWVB），和镰刀病毒（SBV）。因此,之前描述的检测方法在计算机模拟中重新评估其特异性和包容性.基于这一评价,选择的方法进行了修改，或新的设计和测试在双重RT-qPCR反应。检测限（LODs），评估了多路复用对蜜蜂和蜂巢碎片中的敏感性和病毒RNA定量潜力的影响。与先前描述的测试和单个PCR反应中的两种病毒病原体相比，这项研究提供了能够检测到数量增加的病毒变体的诊断测定。
