Diagnosis of Potato Leafroll Virus with Digoxigenin-labeled cRNA Probes

Digoxigenin으로 표지된 cRNA 프로브를 이용한 감자잎말림바이러스(PLRV)의 짐단

  • 서효원 (고령지농업시험장 감자과) ;
  • 함영일 (고령지농업시험장 감자과) ;
  • 오승은 (건국대학교 생물학과) ;
  • 신관용 (고령지농업시험장 감자과) ;
  • 최장경 (강원대학교 농생물학과)
  • Published : 1998.12.01

Abstract

Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

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