Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Abstract

Aims: The purpose of this study was to establish a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. Methods and Results: A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65°C for 60 min. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10−5 dilution level (equivalent to 17·2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. Conclusions: The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. Significance and Impact of the Study: This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow‐out pond, to prevent the disease outbreak.

Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

“…In most cases, the sensitivity of a monoplex LAMP assay, such as LAMP-LFD for V. harveyi detection in added shrimp samples (5 CFU per reaction: Thongkao et al 2015), conventional LAMP for V. harveyi in spiked shellfish cultures (17.2 CFU per reaction: Cao et al 2010), and V. cholera in added shrimp samples (20 CFU per reaction: Srisuk et al 2010) was greater than that of dLAMP-LFD in our study. The lower sensitivity may be due to competition among the number of primers that were used under multidetection conditions, as previously described (Ali and Reynolds 2000;Pang et al 2002;Xie et al 2006;Rashid et al 2009).…”

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

“…2010) and approx. 5–7 times higher than that of LAMP assay for Vibrio harveyi in added shellfish cultures (17·2 CFU per reaction; Cao et al. 2010) and V. cholerae in spiked shrimp sample (20 CFU per reaction; Srisuk et al.…”

Bacterial diseases in marine bivalves

“…In addition, for V. harveyi-related species, highly similar genomes and genome plasticity may also limit precise identification by molecular techniques in some cases (Thompson and Swings, 2006;Sawabe et al, 2007). Thompson et al, 2007;Cano-Gomez et al, 2011), loop-mediated isothermal amplification (LAMP) (Cao et al, 2010), and TaqMan PCR to detect potentially virulent strains for H. tuberculata (Schikorski et al, 2014) (Table 2).…”