Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri
- 作者：Hung-Yueh Yeh ; Craig A. Shoemaker and Phillip H. Klesius
- 关键词：Edwardsiella ictaluri ; Ictalurus punctatus ; LAMP
- 刊名：Journal of Microbiological Methods
- 出版年：2005
- 期刊代码：59_01677012
- 类别：imm
- 出版时间：2005
- 卷：63
- 期：1
- 页码：36-44
- 文件大小：211 K
摘要
Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40–50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of E. ictaluri. A set of four primers, two outer and two inner, was designed specifically to recognize the eip18 gene of this pathogen. The LAMP reaction mix was optimized. Reaction temperature and time of the LAMP assay for the eip18 gene were also optimized at 65 °C for 60 min, respectively. Our results show that the ladder-like pattern of bands sizes from 234 bp specifically to the E. ictaluri gene was amplified. The detection limit of this LAMP assay was about 20 colony forming units. In addition, this optimized LAMP assay was used to detect the E. ictaluri eip18 gene in brains of experimentally challenged channel catfish. Thus, we concluded that the LAMP assay can potentially be used for rapid diagnosis in hatcheries and ponds.