Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus

Abstract

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens

“…Reverse‐transcription (RT) loop‐mediated isothermal amplification (LAMP) reactions were carried out to confirm the identity of FHMNV according to the method developed by Zhang et al. () using three pairs of RT‐LAMP primers (Table ). For GSRV confirmation, we used a set of primers targeting the S10 segment of GSRV developed in our laboratory (Table ).…”

Nidoviruses of Fish and Crustaceans

“…Sequence analysis of the ORF2 gene region amplified by PCR has provided a means of differentiating FHMNV strains isolated in cell culture from FHMs being reared in Minnesota, Wisconsin and Illinois as well as present in jack pike (Esox masquinongy) fed live FHMs in Nebraska and a batch of creek chub (Semotilus atromaculatus) (Batts et al, 2012;McCann, 2012). A constant-temperature RT loop-mediated isothermal amplification (RT-LAMP) test with a sensitivity of ~5 RNA copies has also been developed for FHMNV diagnosis and surveillance (Zhang et al, 2014). While no molecular tests have been reported specifically for WBV diagnosis, immunoblotting using a polyclonal rabbit antiserum to a fusion protein to the pp1a main proteinase (Mpro) motif expressed in bacteria has been used to detect predicted Mpro cleavage products of pp1a and pp1ab in WBVinfected fish cells (Ulferts et al, 2011).…”

Nidoviruses in Aquatic Organisms - Paradigm of a Nascent Concern

“…For diagnosis and surveillance of this pathogen, the development of PCR procedure for FHMNV was accomplished in 2012 [10]. Subsequently, RT-LAMP test with a sensitivity of around 5 copies of RNA [29] and reverse transcriptase PCR assay [26] were standardized for precise diagnosis.…”