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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

Abarshi, M.M., Mohammed, I.U., Wasswa, P., Hillocks, R.J., Holt, J., Legg, J.P., Seal, S.E. ORCID: 0000-0002-3952-1562 and Maruthi, M.N. (2009) 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, 163 (2). pp. 353-359. ISSN 0166-0934 (doi:10.1016/j.jviromet.2009.10.023)

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Abstract

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.

Item Type: Article
Uncontrolled Keywords: CBSV, RT-PCR, diagnosis, cost-effective protocols
Subjects: S Agriculture > S Agriculture (General)
S Agriculture > SB Plant culture
Faculty / Department / Research Group: Faculty of Engineering & Science > Natural Resources Institute
Faculty of Engineering & Science > Natural Resources Institute > Agriculture, Health & Environment Department
Related URLs:
Last Modified: 20 Nov 2014 18:05
Selected for GREAT 2016: None
Selected for GREAT 2017: None
Selected for GREAT 2018: None
URI: http://gala.gre.ac.uk/id/eprint/3361

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