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
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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DOI：
10.1016/j.jviromet.2006.11.021
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发表时间：
2007-04-01
影响因子：
3.1
通讯作者：
Faucon, Jean Paul
中科院分区：
文献类型：
--
作者：
Blanchard, Philippe;Ribiere, Magali;Faucon, Jean Paul
建立了一种基于TaqMan荧光探针（FAM-TAMRA）的两步法实时荧光定量RT-PCR检测蜜蜂慢性蜜蜂麻痹病毒（CBPV）基因组的方法。从CBPV对照RNA和从含有CBPV部分序列的质粒获得的标准曲线表明，该测定提供了在7-log范围内的线性检测（R-2 > 0.99），检测限为100个拷贝，以及可靠的测定间和测定内重现性。CBPV TaqMan方法首先通过定量CBPV基因组负载来评估，所述CBPV基因组负载来自通过局部施用获得的实验感染的蜜蜂样品。发现昆虫身体每个部分（头、胸和腹部）的CBPV拷贝数高达1.9 x 10（10），而在解剖器官（如下颌腺和下咽腺、脑和消化道）中检测到较低的CBPV基因组负荷（高达7.2 x 10（6）CBPV拷贝数）。然后定量来自蜂巢的不同类别的蜜蜂中的CBPV基因组负荷，所述蜜蜂呈现慢性麻痹的典型颤抖症状。在守卫蜂、症状蜂和死亡蜂中发现的CBPV负荷（高达1.9 x 10（13）CBPV拷贝）显著高于觅食蜂、雄蜂和家蜂（高达3.4 x 10（6）CBPV拷贝）。从有症状或死亡的蜜蜂获得的结果支持高CBPV基因组载量与病理表达之间的相关性。此外，高CBPV基因组载量揭示了在守卫蜜蜂中，这类蜜蜂在CBPV感染中可能发挥的关键作用。(c)2006 Elsevier B. V.保留所有权利。
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 1.9 x 10(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 7.2 x 10(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 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(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. (c) 2006 Elsevier B.V. All rights reserved.