Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 112, Issue 1-2, Pages 153-156Publisher
ELSEVIER
DOI: 10.1016/S0166-0934(03)00188-5
Keywords
Citrus yellow mosaic virus; PCR; DNA isolationwoody plant
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Citrus yellow mosaic virus (CYMV), a non-enveloped bacilliform DNA virus causes a severe mosaic disease in sweet oranges in India. CYMV is weakly immunogenic, thus serodiagnosis is not a preferred method for its detection. As an alternative a rapid and reliable detection protocol by polymerase chain reaction (PCR) was developed. However, high levels of polyphenolics and tannins in citrus leaves generally interfered with obtaining good quality DNA, and thus affected the reliable detection of virus by PCR. Consequently, we evaluated the addition of sodium sulphite to a DNA extraction protocol used previously and compared the two methods with a commercially available plant DNeasy Kit (Qiagen). The addition of sodium sulphite improved the yield, quality and stability of DNA. The CYMV DNA was not only amplified at lower template DNA concentration, but also provided better DNA yields. In addition, the sodium sulphite extracted DNA survived at various temperatures much longer than those extracted without addition of sodium sulphite or with the commercial kit. The amplified product of CYMV DNA was cloned, sequenced and found to have 89% sequence identity with the only other sequenced Indian isolate of CYMV. (C) 2003 Elsevier B.V. All rights reserved.
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