Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed.

Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...