title: An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification.
abstract: BACKGROUND
  Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.
  
  
  RESULTS
  A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.
  
  
  CONCLUSIONS
  This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.
authors.affiliation: Institute of Zoology, Fish Biology and Fish Diseases, Faculty of Veterinary Medicine, University of Munich, Germany. soliman@zoofisch.vetmed.uni-muenchen.de
authors.forename: Hatem
authors.initials: H
authors.lastname: Soliman
authors.forename: Mansour
authors.initials: M
authors.lastname: El-Matbouli
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D052160:Benzothiazoles; D002347:Carps / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004259:DNA-Directed DNA Polymerase