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Diseases of Aquatic Organisms

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DAO 73:103-111 (2006)  -  doi:10.3354/dao073103

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay

Ping-Hua Teng1,2, Pei-Yu Lee3, Fu-Chun Lee2, Hung-Wen Chien2, Man-Shu Chen2, Ping-Feng Sung2, Chen Su2, Bor-Rung Ou1,*

1Department of Animal Science and Biotechnology, Tunghai University, Taichung 40704, Taiwan, ROC
2Farming IntelliGene Technical Corporation, Taichung 40755, Taiwan, ROC
3Institute of Medical Biotechnology, Central Taiwan University of Science and Technology, Taichung 406, Taiwan, ROC
*Corresponding author. Email:

ABSTRACT: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus that causes developmental and growth abnormalities in Pacific white shrimp Litopenaeus vannamei (also known as Penaeus vannamei). Nucleic acid based methods such as in situ hybridization (ISH) and PCR have been commonly used for IHHNV detection. Ramification amplification (RAM), an isothermal nucleic acid amplification approach, was used in this study to detect IHHNV in L. vannamei. RAM offers many advantages over PCR, including simple procedures and short detection time, and is labor-saving and cost-effective. RAM exponentially amplifies a circular oligonucleotide amplicon (C probe) after a target-specific ligation step through sequential primer extension and strand displacement processes. The conditions of an IHHNV RAM assay were optimized using artificial templates and targets prior to application. Using DNA of IHHNV-infected L. vannamei as targets, results revealed that RAM amplified target DNA with similar sensitivity as PCR. RAM offers competitive levels of speed, simplicity and sensitivity among various pathogen diagnostic methods.

KEY WORDS: IHHNV · Litopenaeus vannamei · Ramification amplification · Isothermal amplification

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