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Aims:  The aim of this paper was to develop a loop‐mediated isothermal amplification (LAMP) method for rapid, sensitive and inexpensive detection of Singapore grouper iridovirus (SGIV) in grouper (GP), Epinephelus sp. Methods and Results:  A set of six specific primers was designed by targeting the SGIV ORF‐014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65°C in a simple water bath. The detection limit is about 0·02 fg (equivalent to 6·3 copies) of plasmid ORF‐014L. LAMP products could be judged with three different methods. There were no cross‐reactions with seven other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus‐infected GP cells and GP tissues effectively. Conclusions:  The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for the detection of SGIV in cells and in GP tissues. Significance and Impact of the Study:  The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.

BackgroundHepatitis C virus (HCV) is a human pathogen causing chronic liver disease in about 200 million people worldwide. However, HCV resistance to interferon treatment is one of the important clinical implications, suggesting the necessity to seek new therapies. It has already been shown that some forms of the catalytic RNA moiety from E. coli RNase P, M1 RNA, can be introduced into the cytoplasm of mammalian cells for the purpose of carrying out targeted cleavage of mRNA molecules. Our study is to use an engineering M1 RNA (i.e. M1GS) for inhibiting HCV replication and demonstrates the utility of this ribozyme for antiviral applications.ResultsBy analyzing the sequence and structure of the 5′ untranslated region of HCV RNA, a putative cleavage site (C67-G68) was selected for ribozyme designing. Based on the flanking sequence of this site, a targeting M1GS ribozyme (M1GS-HCV/C67) was constructed by linking a custom guide sequence (GS) to the 3′ termini of catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coli through an 88 nt-long bridge sequence. In vitro cleavage assays confirmed that the engineered M1GS ribozyme cleaved the targeted RNA specifically. Moreover, ~85% reduction in the expression levels of HCV proteins and >1000-fold reduction in viral growth were observed in supernatant of cultured cells that transfected the functional ribozyme. In contrast, the HCV core expression and viral growth were not significantly affected by a “disabled” ribozyme (i.e. M1GS-HCV/C67*). Moreover, cholesterol-conjugated M1GS ribozyme (i.e. Chol-M1GS-HCV/C67) showed almost the same bioactivities with M1GS-HCV/C67, demonstrating the potential to improve in vivo pharmacokinetic properties of M1GS-based RNA therapeutics.ConclusionOur results provide direct evidence that the M1GS ribozyme can function as an antiviral agent and effectively inhibit gene expression and multiplication of HCV.