The analysis of DNA polymerase gene of abalone herpesvirus Taiwan isolate and its application on the diagnosis
Date Issued
2014
Date
2014
Author(s)
Chen, Min-Hsiu
Abstract
A 5781-base pair (bp) fragment of genomic DNA was obtained from Taiwanese abalone herpesvirus and showed 99% (5767/5779) identity in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Identity of the amino acid sequence was 30% (563/1856) with the DNA polymerase of ostreid herpesvirus 1. In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606 bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic method for herpesvirus infections in abalone. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized at 63℃ and 60 min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus.
Subjects
疱疹病毒
九孔鮑
DNA聚合酶
聚合酶鏈反應 (PCR)
恆溫環形核酸增幅法 (LAMP)
SYBR green即時聚合酶鏈反應
Type
thesis
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