关键词: 苹果茎痘病毒, TaqMan 探针, 实时荧光定量RT-PCR

Abstract: This study established a TaqMan real-time RT-PCR method for detecting Apple stem pitting virus（ASPV）. A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASPV coat protein gene（cp）. The RNA standard of ASPV was prepared with transcription in vitro and the standard curve was plotted. The specificity，sensitivity and reproducibility of this method were evaluated. The results showed that the coefficient of determination（R2）was 0.996 and the amplification efficiency（E）was 99%. There was no crossing reaction with Apple stem grooving virus（ASGV），Apple chlorotic leaf spot virus（ACLSV）and Apple scar skin viroid（ASSVd），indicating a strong specificity. The detection limit of this method was 1.31 × 102 copies ? μL-1 and the sensitivity was 100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1.85%. This method could be used to detect ASPV rapidly and quantitatively in field samples with strong specificity，high sensitivity and reliable reproducibility.

Key words: Apple stem pitting virus（ASPV）, TaqMan probe, real-time RT-PCR