Rapid, sensitive and simple detection method for koi herpesvirus using loop-mediated isothermal amplification.
New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI-5 PCR amplicon (SphI-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65 degrees C for 60 min. The detection limit of both LAMP was six copies, equal to the modified SphI-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. SphI-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.
Yoshino
Manabu
M
Biochemical Research Laboratory, Eiken Chemical Co., Ltd, Tochigi 329-0114, Japan. manabu_yoshino@eiken.co.jp
Watari
Hajime
H
Kojima
Tadashi
T
Ikedo
Masanari
M
Kurita
Jun
J
D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific; D011756:Diphosphates; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D005880:Gills / Q000821:virology; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D017616:Magnesium Compounds; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D011756:Diphosphates; D017616:Magnesium Compounds; C034732:magnesium pyrophosphate; C046184:endodeoxyribonuclease PaeI; D015252:Deoxyribonucleases, Type II Site-Specific