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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus
Journal of Virological Methods, Volume 163, No. 2, Year 2010
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Description
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 10× cheaper (£0.53 = US$0.80 per sample) than the RNeasy method (£5.91 = US$8.86 per sample). A two-step RT-PCR (£1.34 = US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (£4.48 = 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. © 2009 Elsevier B.V.
Authors & Co-Authors
Abarshi, Muawiya Musa
United Kingdom, Chatham
Natural Resources Institute
Mohammed, Ibrahim U.
United Kingdom, Chatham
Natural Resources Institute
Wasswa, Peter
United Kingdom, Chatham
Natural Resources Institute
Hillocks, Rory J.
United Kingdom, Chatham
Natural Resources Institute
Holt, Johnson
United Kingdom, Chatham
Natural Resources Institute
Legg, James P.
Tanzania, Dar es Salaam
International Institute of Tropical Agriculture Iita
Seal, Susan E.
United Kingdom, Chatham
Natural Resources Institute
Maruthi, Midatharahally Narasegowda
United Kingdom, Chatham
Natural Resources Institute
Statistics
Citations: 96
Authors: 8
Affiliations: 2
Identifiers
Doi:
10.1016/j.jviromet.2009.10.023
ISSN:
01660934
Study Design
Cohort Study