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首页> 外文期刊>Journal of Virological Methods >Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony
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Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony

机译：实时两步法RT-PCR测定定量评估实验感染的蜜蜂组织和有症状菌落生命阶段中的慢性蜜蜂麻痹病毒（CBPV）基因组的评估

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A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9x10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2x10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9x10(13) CBPV copies) than in forager, drones and house bees (up to 3.4x10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.
机译：基于TaqMan技术，使用荧光探针（FAM-TAMRA）进行了两步实时RT-PCR测定，以定量蜜蜂样品中的慢性蜜蜂麻痹病毒（CBPV）基因组。从CBPV对照RNA和包含CBPV部分序列的质粒获得的标准曲线表明，该测定法可在7个对数范围（R（2）> 0.99）内提供线性检测，检测限为100拷贝，并且可靠批间和批内重复性。还验证了包括RNA纯化和cDNA合成在内的标准化。首先通过量化来自局部应用获得的实验性感染的蜜蜂样品中的CBPV基因组负荷来评估CBPV TaqMan方法。每个昆虫体（头部，胸部和腹部）每个部分的CBPV拷贝数高达1.9x10（10），而在解剖器官（如下颌和下咽腺，脑和消化道）中检测到较低的CBPV基因组负荷（高达7.2x10） （6）CBPV副本）。然后对蜂巢中表现出典型的慢性麻痹症状的蜂巢中不同类别蜜蜂的CBPV基因组负荷进行了定量。在警卫，有症状和死蜂（最多1.9x10（13）CBPV副本）中发现的CBPV负载比在觅食者，无人机和家养蜜蜂（最多3.4x10（6）CBPV副本）中更高。有症状或死蜜蜂的结果支持高CBPV基因组负荷与病理表达之间的相关性。此外，在保护蜂中发现的高CBPV基因组负荷凸显了这类蜜蜂在CBPV感染中可能发挥的关键作用。

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