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| An Efficient TaqMan Quantitative Real-time PCR(qRT-PCR) Assay for Detecting Apple stem grooving virus (ASGV) |
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Abstract This study aimed at establishing a TaqMan quantitative Real-time RT-PCR (qRT-PCR) assay for detecting Apple stem grooving virus (ASGV). A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASGV coat protein (CP) gene. The recombinant plasmid of ASGV-cp was constructed as positive standard to generate standard curve, and the specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient (R2) of standard curve was 0.999 and the amplification efficiency (E) was 96.8%. There was no crossing reaction with Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), indicating a strong specificity. The sensitivity of this method was 10 copies/μL which was 1 000 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1%. This method could be used to detect ASGV rapidly in practical samples with strong specificity, high sensitivity and reliable reproducibility.
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Received: 10 December 2014
Published: 13 April 2015
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