微量板杂交法和斑点杂交法检测菊花矮化类病毒的比较

Comparison Between Microplate Hybridization Method and Dot Blot Method for the Detection of Chrysanthemum Stunt Viroid

利用狄高辛(DIG)标记制备菊花矮化类病毒(CSV)特异性探针,由反转录-聚合酶链反应(RT-PCR)进行CSV RNA的反转录和cDNA的扩增,然后比较2种核酸杂交方法——微量板杂交和斑点杂交检测CSV的灵敏度。本研究结果表明,30 mg干燥样本抽提的CSV RNA被稀释400倍,取4 μL(约750 pg样本抽提的RNA)经RT-PCR,其产物再用10×SSC稀释100倍,采用微量板杂交仍可以很容易得到阳性结果,而采用斑点杂交法,同样条件下PCR产物被稀释8倍就不易得到阳性结果。所以,微量板杂交法比斑点杂交法具更高的灵敏度。

The specific probe of chrysanthmum stunt viroid (CSV) was prepared by using digoxigenin (DIG)-labelled. Amplification of cDNA to viral RNA sequence was carried out with reverse transcription and polymerase chain reaction (RT-PCR). Then the sensitivity of two nucleic acid hybridization methods-microplate hybridization and dot blot was compared. The results showed that after CSV RNA extracted from 30 mg sample was diluted 400 times and the RT-PCR products from 4 μL extraction were diluted 100 times with 10×SSC, it was still easy to obtain positive result when the diluted RT-PCR products were detected with microplate hybridization, but it was difficult to detect the RT-PCR products diluted 8 times with dot blot in the same conditions. Therefore microplate hybridization method is more sensitive than dot blot method.