使用[32P]RNA探针进行马铃薯纺锤块茎类病毒的斑点杂交检测。

Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid.

作者信息

Lakshman D K, Hiruki C, Wu X N, Leung W C

出版信息

J Virol Methods. 1986 Nov;14(3-4):309-19. doi: 10.1016/0166-0934(86)90033-9.

Abstract

A dot-hybridization assay using 32P-labelled RNA probes (+RNA and cRNA) transcribed from potato spindle tuber viroid (PSTV) cDNA was described. A complete cDNA copy of PSTV, originally cloned in pBR 322 (pAV 401) was subcloned in the BamHI site of a 'Riboprobe' cloning vectors pSP 64 and pSP 65 in opposite orientations. The reconstructed plasmids were designated pDX 1 and pDX 4, respectively. Transcription of pDX 1 and pDX 4 plasmids by SP6 RNA polymerase resulted in the generation of PSTV-specific RNA (+RNA) and PSTV complementary RNA (cRNA), respectively. The cRNA probe was much more sensitive than the +RNA probe and the nick-translated cDNA probe from the plasmid pAV 401 for the detection of PSTV in clarified plant sap. As little as 1.4 pg of purified PSTV mixed in clarified sap from uninoculated tomato leaves has been detected using cRNA probe. A relatively simple procedure using cetyltrimethyl ammonium bromide (CTAB) as nucleic acid precipitant and an enrichment step for the purification of PSTV was described.

摘要

本文描述了一种斑点杂交试验，该试验使用从马铃薯纺锤块茎类病毒（PSTV）cDNA转录而来的32P标记的RNA探针（+RNA和cRNA）。最初克隆于pBR 322（pAV 401）的PSTV完整cDNA拷贝，以相反方向亚克隆到“Riboprobe”克隆载体pSP 64和pSP 65的BamHI位点。重建后的质粒分别命名为pDX 1和pDX 4。用SP6 RNA聚合酶转录pDX 1和pDX 4质粒，分别产生PSTV特异性RNA（+RNA）和PSTV互补RNA（cRNA）。在检测澄清植物汁液中的PSTV时，cRNA探针比+RNA探针和来自质粒pAV 401的切口平移cDNA探针灵敏得多。使用cRNA探针，已检测到在未接种番茄叶片的澄清汁液中混入低至1.4 pg的纯化PSTV。本文还描述了一种相对简单的方法，该方法使用十六烷基三甲基溴化铵（CTAB）作为核酸沉淀剂，并采用富集步骤来纯化PSTV。