Detection of Papaya ringspot virus (PRSV) by cDNA Probes Labelled with Digoxgenin Labelling and AlkPhos Direct Labelling
Detection of Papaya ringspot virus (PRSV) by cDNA Probes Labelled with Digoxgenin Labelling and AlkPhos Direct Labelling
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发表时间：
2007
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通讯作者：
F. Hui
中科院分区：
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作者：
F. Hui
根据GenBank公布的中国Sm株PRSV(番木瓜环斑病毒)CP基因序列，采用特异性引物进行RT-PCR。使用从PRSV感染的番木瓜梅中红2号中提取的总RNA，将克隆的cDNA连接至pGEM-T easy质粒载体并测序。以重组质粒为模板，用 DIG PCR 探针合成试剂盒进行标记。或者，还使用AlkPhos直接标记方法来标记从电泳中回收的cDNA。结果表明，梅中红2号分离株序列与中国PRSV-Sm基因组序列一致性为94.7%； DIG标记的cDNA探针（861、455和215 bp）检测PRSV时产生相同的杂交结果，与RT-PCR结果一致，且861 bp探针获得最清晰的杂交信号，核酸杂交的灵敏度和特异性可以满足传统诊断的需要； AlkPhos直接标记的861 bp探针可以识别PRSV，而455 bp则无效；叶脉中也进行了DIG标记探针，得到了与RT-PCR相同的结果。
RT- PCR was carried out with specific primers according to published sequences of PRSV(Papaya ringspot virus) CP genes of Chinese Sm strain in GenBank. Using total RNA extracted from PRSV-infected Meizhonghong No.2 of Carica papaya L. the cloned cDNA was ligated to pGEM-T easy plasmid vector and sequenced. Recombinant plasmids were used as the template for labelling with DIG PCR Probe Synthesis Kit. Alternatively, AlkPhos direct labelling method was also used to label cDNA recovered from electrophoresis. The results indicated that the sequence of the Meizhonghong No.2 isolate showed 94.7 % identity with the Chinese PRSV- Sm genome; The cDNA probes (861, 455 and 215 bp) labelled by DIG produced the identical hybridization result for detecting the PRSV, which were in agreement with that of RT- PCR, and the clearest hybridization signal was obtained with 861 bp probes, and sensitivity and speciality of nucleic acid hybridization can satisfy the needs of traditional diagnosis; The 861 bp probe from AlkPhos direct labelling could identify PRSV while the 455 bp one was invalid; and DIG labelling probe was also performed in leaf vein and the same result as using RT-PCR was obtained.