Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper.
Isolation of intact high quality RNA suitable for RT-PCR from black pepper is greatly hindered by the presence of polyphenols and polysaccharides. These compounds adversely affect the sensitivity of virus detection by RT-PCR. The present study evaluated the effect of sodium sulphite in enhancing RNA yield and quality in a modified acid guanidium thiocyanate-phenol-chloroform (AGPC) protocol. The results were compared with the standard AGPC method and RNeasy Plant Mini Kit (Qiagen) for detection of Cucumber mosaic virus through RT-PCR. The addition of sodium sulphite in the extraction buffer increased the sensitivity of virus detection. Higher sensitivity of detection (than obtained from the kit) was seen when sodium sulphite was used at 0.5%. Similar levels of sensitivity were also observed for the detection of Cucumber mosaic virus from Piper longum.
Siju
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Division of Crop Protection, Indian Institute of Spices Research, Calicut 673012, Kerala, India.
Madhubala
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Bhat
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AI
D017799:Cucumovirus / Q000235:genetics / Q000302:isolation & purification*; D029222:Piper nigrum / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000494:pharmacology*
D012367:RNA, Viral; D013447:Sulfites; C025026:sodium sulfite