RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis

Sweet potato virus disease (SPVD), which causes severe crop losses in Africa, is caused by a complex of sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Extraction of pure RNA (for diagnosis by reverse transcriptase polymerase chain reaction [RT-PCR...

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Bibliographic Details
Published in:Molecular biotechnology 1998-10, Vol.10 (2), p.187-190
Main Authors: Fenby, N.S. (Bristol University, Bristol, UK.), Seal, S, Gibson, R.W, Foster, G.D
Format: Article
Language:English
Subjects:
Agricultural biotechnology
Amplification
Biological and medical sciences
Biotechnology
Crinivirus
Crop diseases
Deoxyribonucleic acid
DNA
Extraction procedures
Fundamental and applied biological sciences. Psychology
IPOMOEA BATATAS
Methods. Procedures. Technologies
Others
Plant Diseases - genetics
Plant Diseases - virology
PLANT VIRUSES
Polymerase chain reaction
Potatoes
POTYVIRUS
Potyvirus - genetics
POTYVIRUSES
Resins, Plant - pharmacology
Reverse Transcriptase Polymerase Chain Reaction - drug effects
Ribonucleic acid
RNA
RNA viruses
RNA, Plant - drug effects
RNA, Plant - genetics
RNA, Plant - isolation & purification
RNA, Viral - drug effects
RNA, Viral - genetics
RNA, Viral - isolation & purification
RNA-directed DNA polymerase
Solanaceae - genetics
Solanaceae - virology
Solanum tuberosum
SWEET POTATO CHLOROTIC STUNT CONIVIRUS
sweet potato chlorotic stunt virus
SWEET POTATO FEATHERY MOTTLE POTYVIRUS
Sweet potato feathery mottle virus
sweet potato virus disease
Sweet potatoes
Various methods and equipments
Vegetables
VIRUS DE LAS PLANTAS
VIRUS DES VEGETAUX
Viruses
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Description
Summary:Sweet potato virus disease (SPVD), which causes severe crop losses in Africa, is caused by a complex of sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Extraction of pure RNA (for diagnosis by reverse transcriptase polymerase chain reaction [RT-PCR] methods) from diseased sweet potato proved to be difficult using five different total-RNA extraction procedures: (a) that of Chomczynski and Sacchi (1) (b) an adaptation of Lodhi et al., (2) and three commercially available kits (c) Clonsep (Clontech, Clontech Labs Inc, Palo Alto, CA; (d) RNeasy (Qiagen, West Sussex, UK); and (e) RNA isolator (Genosys Biotechnologies). Four of these methods (b-e) generated sufficient RNA, but it was unsuitable for RT-PCR amplification of SPFMV. When these RNA samples were treated with Wizard DNA extraction resin (Promega), the inhibitors of RT-PCR were consistently removed from three (b-d) of these four samples. Another deproteinating step was needed to allow RT-PCR amplification of sample e. The Wizard DNA column purification in conjunction with one of these total RNA extraction methods facilitates quick and reliable extraction of pure RNA for diagnostic purposes and might be suitable for similar problematic plant material.
ISSN:1073-6085
1559-0305
DOI:10.1007/BF02760865