A novel method of real-time reverse-transcription loop-mediated isothermal amplification developed for rapid and quantitative detection of a new genotype (YHV-8) of yellow head virus

Abstract

A new genotype of yellow head virus (YHV), designated as YHV-8, was found in farmed shrimp Fenneropenaeus chinensis suffering suspectedly from EMS/AHPNS (early mortality disease/acute hepatopancreatic necrosis disease) in China in 2012. In this study, a one-step, real-time reverse-transcription loop-mediated isothermal amplification (rRT-LAMP) assay was developed for better detection of both genotypes of YHV-1 and YHV-8. A set of six specific primers was successfully designed targeting a conserved region of the YHV genome. The LAMP reaction was optimized to contain 8mmoll(-1) Mg(2+) and 1·4mmoll(-1) dNTPs, and to be performed at 58°C for 60min. The detection sensitivity of the rRT-LAMP method was as low as 7×10(0) copies per reaction. The specificity of the method was validated by the absence of any cross-reaction with the RNA samples extracted from other shrimp viruses (Taura syndrome virus, white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus) and specific pathogen-free (SPF) shrimp. The resulting standard curves showed high correlation coefficient values. Furthermore, the test of farm samples showed that YHV was detected in three of 111 Litopenaeus vannamei, six of eight Fenneropenaeus chinensis, five of 19 Macrobrachium rosenbergii and none of the nine Marsupenaeus japonicus. These results suggest that this assay is applicable widely as a new rapid and sensitive detection method in the research of YHV. In this study, we designate a new genotype of yellow head virus (YHV) as YHV genotype 8 (YHV-8) which was detected in diseased shrimp in China. A rapid, sensitive and specific rRT-LAMP detecting method for both YHV-8 and YHV-1 has been established. It is anticipated that this novel assay will be instrumental for diagnosis and surveillance of the virulent genotypes of YHV.