以非放射性物质地高辛为标记物,采用随机引物标记法获得了灵敏度高、特异性强的DNA探针,通过优化杂交时间以及上样量,建立了桑树皱叶病毒(mulberry crinkle leaf virus,MCLV)的核酸斑点杂交(nucleic acid spot hybridization,NASH)检测体系。与PCR检测方法相比,建立的NASH检测体系准确率达93.1%。利用建立的NASH技术对江苏省镇江市3个不同地块(Ⅰ、Ⅱ、Ⅲ)采集的60个桑叶样品进行检测以分析MCLV病毒在该地区的分布情况及与桑树病毒样病害的关系,结果显示,Ⅰ地块检出率20%,Ⅱ地块检出率90%,Ⅲ地块检出率95%,平均检出率68.3%,说明MCLV在该地区分布广泛;另外,在某些无任何病毒病症状的植株上检出MCLV,而在某些有明显花叶或萎缩症状的植株上未检测出MCLV,说明MCLV可能与桑树的花叶或萎缩症状无明显关联。
DNA probes specific and sensitive to mulberry crinkle leaf virus (MCLV)were labeled with non-radioactive digoxigenin (DIG)and were generated according to the random-primed DNA labeling method.A nucleic acid spot hybridization (NASH)method was developed using the DIG-labeled DNA probes to detect MCLV by optimizing the hybridization time and loading volumes.The diagnosis accuracy of the established MCLV-specific NASH was 93.1% compared to PCR.Sixty mulberry leaf samples from three different mulberry fields (Ⅰ,Ⅱ,Ⅲ )in Zhenjiang,Jiangsu were collected and detected by NASH to investigate the distribution of MCLV in this area and the relationship between the MCLV and mulberry disease with virus-like symptoms.The detection rate of MCLV in mulberry leaves from field Ⅰ,Ⅱ and Ⅲ was 20%,90%and 95%,respectively,with an average of 68.3%,indicating that MCLV is widely distributed in this area.The results also showed that no MCLV was detected in leaf samples from the mulberry trees with obvious mosaic or dwarf symptoms,while MCLV was detected in leaf samples from the mulberry trees without any virus-like symptoms,suggesting that MCLV might not be responsible for mosaic or dwarf symptoms exhibiting on the mulberry trees.
Acta Sericologica Sinica
Mulberry crinkle leaf virus (MCLV)
Nucleic acid spot hybridization (NASH)