Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus
Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus
复制标题
DOI：
10.1016/j.jviromet.2009.10.023
复制
发表时间：
2010-02-01
影响因子：
3.1
通讯作者：
Maruthi, M. N.
中科院分区：
文献类型：
--
作者：
Abarshi, M. M.;Mohammed, I. U.;Maruthi, M. N.
为准确诊断木薯褐条病毒（CBSV），对木薯褐条病毒的取样程序和诊断方法进行了优化。十六烷基三甲基溴化铵（CTAB）的方法进行了优化，从受感染的木薯植物的样品制备和RNeasy植物迷你试剂盒（凯杰）的灵敏度，再现性和成本进行了比较。CBSV在使用任一方法提取的总RNA中易于检测。这两种方法之间的主要区别在于消耗品的成本，CTAB比RNeasy方法便宜10倍（每个样品0.53英镑= 0.80美元）（每个样品5.91磅= 8.86美元）。两步RT-PCR（每个样品1.34磅= 2.01美元）虽然灵敏度较低，但至少比一步RT-PCR（4.48磅= 6.72美元）便宜3倍。两种RT-PCR检测结果一致表明，CBSV在症状叶片和无症状叶片中均存在，表明无症状叶片可用于病毒诊断。根据所需的准确性，每个田地取样100-400株植物是CBSD诊断的适当建议，分别给出检测6.7- 1.7%的疾病发病率的99.9%的概率。在复合采样中，在10（-4）倍稀释度下检测到CBSV，表明对许多CBSV样本进行索引的最有效方法是筛选合并样本。以下描述的诊断方案是目前检测CBSV的可靠且最具成本效益的方法。(C)2009爱思唯尔有限公司版权所有。
Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10x cheaper (0.53=US pound$0.80 per sample) than the RNeasy method (5.91 pound = US$8.86 per sample). A two-step RT-PCR (1.34 pound = US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (4.48 pound = US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100-400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7-1.7%, respectively. CBSV was detected at 10(-4)-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV. (C) 2009 Elsevier B.V. All rights reserved.