title: 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.
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao, Shandong 266071, China; Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Isaac
authors.initials: I
authors.lastname: Standish
authors.affiliation: Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA; Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Winters
authors.affiliation: La Crosse Fish Health Laboratory, United States Fish and Wildlife Service, 555 Lester Avenue, Onalaska, WI 54650, USA.
authors.forename: Corey
authors.initials: C
authors.lastname: Puzach
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom.
authors.forename: Rachel
authors.initials: R
authors.lastname: Ulferts
authors.affiliation: Centre for Infection and Immunity, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom; Institute of Medical Virology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany.
authors.forename: John
authors.initials: J
authors.lastname: Ziebuhr
authors.affiliation: Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA; Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI 48824, USA. Electronic address: faisal@cvm.msu.edu.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Faisal
mesh_terms: D000818:Animals; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D003530:Cyprinidae / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D028381:Nidovirales / Q000235:genetics / Q000302:isolation & purification*; D009656:North America; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D064370:Spike Glycoprotein, Coronavirus / Q000235:genetics; D019562:Viral Load / Q000379:methods*
chemical_list: D017931:DNA Primers; D064370:Spike Glycoprotein, Coronavirus
keywords: Calcein; Fathead minnow nidovirus; Quantitative RT-LAMP; Real-time; Reverse transcription loop-mediated isothermal amplification