Rapid Cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

Abstract

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of one step loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65°C along with detecting the products through visual inspection via hybridization at 65°C for 5 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fg DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article was very sensitive, fast, and reliable diagnostic tools for CyHV-3 detection.