实时荧光RT-PCR一步法检测苹果茎沟病毒

Detection of Apple stem grooving virus by real-time fluorescent RT-PCR one step assay

根据苹果茎沟病毒(ASGV)各分离物外壳蛋白(CP)基因的保守序列,设计并合成1对特异性引物和1条TaqMan-MGB探针,建立了对ASGV的实时荧光RT-PCR检测方法。TaqMan-MGB探针3’端具有小沟结合分子(Minor groove binder, MGB),提高了探针的Tm值,缩短了探针长度,解决了病毒各分离物之间基因组变异较大、难以找到较长一致的序列来设计探针的困难。该方法的检测灵敏度比常规RT-PCR电泳检测高约10倍,对于ASGV等在果树体内含量较低的病毒尤为适用。此方法快速、灵敏,整个检测过程完全闭管,无需PCR后处理。

A pair of primers and a TaqMan-MGB probe based on the conserved nucleotide sequence of coat protein （CP） gene of several Apple stem grooving virus （ASGV） isolates were designed and synthesized. A novel real-time fluorescent RT-PCR was established to detect ASGV. The covalent attachment of the minor groove binder moiety at the 3′ end of the probe raised the melting temperature to a range suitable for real-time analysis, shortened the probe, and solved the problem of detecting virus with high genome variability among isolates, where an identical sequence can not be found without multiple mismatches. The method was ten times more sensitive than normal RT-PCR and suitable for the detection of those viruses with low concentration in fruit trees. The method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products.