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Development of an overlapping PCR method to clone the full genome of infectious hypodermal and hematopoietic necrosis
Yong-Wei Wei1, Dong-Dong Fan1, Jiong Chen2
1Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
Abstract:
Decapod Penstyldensovirus 1, previously named as infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an economically important pathogen that causes shrimp diseases worldwide. However, a rapid method for cloning full-length IHHNV genome sequences is still lacking, which makes it difficult to study the genomics and molecular epidemiology of IHHNV. Here, a novel and rapid PCR technique was developed to determine the complete genomic sequences of IHHNV. The IHHNV genome was amplified in two overlapping fragments which each yielded a 2kb PCR product covering the first half or the second half of IHHNV genome, respectively. Using this method, six complete genomic sequences of IHHNV, which were collected from different regions of Zhejiang province in China, were cloned and sequenced successfully. The new cloning method will greatly facilitate the study on the genomics and molecular epidemiology of IHHNV.
