Quantitative PCR detection for abalone shriveling syndrome-associated virus

• Published in: Journal of virological methods 2012-09, Vol.184 (1-2), p.15-20
• Main Authors: Jiang, Jing-Zhe, Zhu, Zhen-Ni, Zhang, Han, Liang, Ya-Yu, Guo, Zhi-Xun, Liu, Guang-Feng, Su, You-Lu, Wang, Jiang-Yong
• Format: Article
• Language: English
• Subjects: Abalone; Abalone shriveling syndrome-associated virus; Animals; annealing; Biological and medical sciences; China; coasts; detection limit; DNA Primers - genetics; DNA viruses; DNA Viruses - genetics; DNA Viruses - isolation & purification; Fundamental and applied biological sciences. Psychology; Gastropoda - virology; genes; Haliotis diversicolor; hybrids; juveniles; Microbiology; pathogenicity; plasmids; quantitative polymerase chain reaction; Quantitative real time PCR; Real-Time Polymerase Chain Reaction - methods; seafoods; Sensitivity and Specificity; SYBR Green; Techniques used in virology; temperature; Virology; Virology - methods; viruses; yields
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.04.009

► This is believed to be the first report of a qPCR method for AbSV detection. ► The detection limits was up to 10 virus copies in a 20-μL reaction mixture. ► AbSV was detected in diseased and healthy abalones from the southern coast of China. Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-μL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years.