Molecular Characterization And Real-time Fluorescent RT-PCR Detection Of Apple Stem Grooving Virus

Kuala pear is a famous fruit variety only produced in Kuala in Xinjiang. Because of its particular flavor, it wins a lot of consumers at home and abroad. But, recently, its appearance and color have changed. Its quality has become poorer. All these greatly menace the production and market value of the famous fruit. All these changes were reported to relate to viral infection.Kuala pear virus isolate (KRL-1) which was originally obtained from Kuala pear's petal by Yejuan DU in April, 2002 was identified as apple stem grooving virus (ASGV) by host range, ELISA, electron microscopy, and RT-PCR. However, KRL-1 was obviously different from other ASGV isolates in biological symptoms, so the coat protein gene (CP) of KRL-1 was cloned and sequenced. The sequence analysis showed that the capsid protein gene of KRL-1 contained 714 nucleotides, encoding a protein of 237 amino acids. The nucleotide (nt) and amino acid (aa) sequence of CP gene of this isolate shared 90% to 93% and 94% to 98% similarities to that of other ASGV isolates, respectively. It is the first time that the complete coat protein gene of ASGV was reported in China except Taiwan, and further molecular detection method can be based on this.ASGV has been recorded from most fruits production areas, and the virus's concentration is low in host trees. There is no obvious symptom in infected tree. It causes great difficulties for early diagnosis and prevention. The existing detection methods easily lead to misdiagnosis. So, a pair of primers and a TaqMan-MGB probe based on the conserved nucleotide sequence of coat protein gene of eight Apple stem grooving virus isolates were designed and synthesized. A novel real-time fluorescent RT-PCR method with TaqMan-MGB probe of 15nt length 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 ASGV with high genome variability among isolates, where an identical sequence could not be found for designing functional normal TaqMan probe. The method was ten times more sensitive than normal RT-PCR, with the dilution limit of 20 pg total RNA in reaction mixture containing 3.5mM of Mg²⁺,0.6μM of either primer and 0.6μM probe, and accurate for the detection of those viruses with low concentration in infected trees. The method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products. It can provide a method for detection ASGV and investigation its incidence in both Kuala pear and disease-free seedlings.