Development of Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cyprinid Herpesvirus-3 (CyHV-3) and its comparison to PCR

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is an etiological agent of an emerging and notifiable disease and one of the mostrisky factors affecting common carp and koi worldwide production with high mortality rates. Molecular methods have been developed and routinely used for the detection of KHV infections. These diagnostic tools are sensitive but still have some drawbacks: they require expensive and bulky apparatus, a large sample volume, and long sample preparation/detection times. Therefore, it is desirable to develop simpler, faster, cheaper and more portable detection methods. Loop-mediated isothermal amplification (LAMP) technique is a novel and next-generation tool which can amplify nucleic acids with high specificity, sensitivity and rapidity under isothermal conditions. In this study, a fragment of the CyHV-3 TK gene was amplified at 65°C in the presence of Bst DNA polymerase and a set of six specially designed primer mixture. The reliability, sensitivity and specificity of this assay for rapid detection of CyHV-3 was investigated and compared to conventional PCR. LAMP assay was successfully developed with the detection of KHV- generated product, and showed 100% specificity and sensitivity of 1.25 fg/µL, that is comparable to the most sensitive method reported to date. The assay was 8000 fold more sensitive and three times faster than conventional PCR. The LAMP assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of CyHV-3..