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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 Resale or republication not permitted without written consent of the publisherDis Aquat Org 73: [103][104][105][106][107][108][109][110][111] 2006 Ramification amplification (RAM), a novel DNA amplification method, was first described in 1998 by Zhang et al. (1998). RAM is an isothermal amplification method that utilizes a circularizable oligodeoxyribonucleotide probe (C probe) as an amplifiable target. The C probe is a synthetic oligonucleotide with a sequence complementary to the specific target sequence. Upon hybridization to the target DNA, the 5' and 3' ends of the C probe are drawn together and can be ligated to form a closed circular molecule (Nilsson et al. 1994). Under isothermal condition, the circular C probe is subsequently amplified with a pair of C probe-specific primers, driven by Bst DNA polymerase with high strand-displacement activity. This amplification is also named hyperbranched rolling circle amplification or cascade rolling circle amplification.After RAM was first demonstrated by Zhang et al. (1998), it became widely applied to DNA-, RNA-or protein-based diagnosis and single nucleotide polymorphism analysis (Barany 1991, Tyagi et al. 1996, Lizardi et al. 1998, Thomas et al. 1999. Other isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have also been reported to detect targets at the fentogram range under isothermal conditions (Kono et al. 2004). However, RAM includes a ligation step that offers better specificity over other isothermal amplification strategies (Zhang et al. 1998). If mismatch occurs at the ends of the C probes at the annealing stage, the ligation step in RAM would not proceed to allow the subsequent signal amplification step. Furthermore, RAM could yield more than 10 6 copies of amplicons within 1 h fr...