Development and application of a loop-mediated isothermal amplification method for rapid detection of Haemophilus parasuis

• Published in: African journal of biotechnology 2011-09, Vol.10 (50), p.10263-10270
• Main Authors: Jian Min, Zhang, Hai Yan, Shen, Cheng Gang, Xu, Li Li, Guo, Bin, Zhang, Jing Yi, Li, Ji Dang, Chen, Hui Ying, Fan, Ming, Liao
• Format: Article
• Language: English
• Subjects: Haemophilus parasuis
• ISSN: 1684-5315; 1684-5315
• DOI: 10.5897/AJB10.1828

Haemophilus parasuis is the causative agent of Glasser's disease that has received much attention recently, due to the increasing economic losses this disease inflicts upon the pig industry worldwide. In this study, loop-mediated isothermal amplification method (LAMP) methodology was designed for diagnosing H. parasuis infections and tested against 56 clinical samples. Two sets of primers for LAMP were designed based on the H. parasuis inf B gene sequence. Target DNA was amplified and visualized on agarose gels after 50 min incubation at 63 degree C. The LAMP amplicon was also directly visualized in the reaction tubes by the naked eye following the addition of SYBR green I. The detection limit of the inf B-LAMP method was 10 cfu mL super(-1), that was 10 times more sensitive than conventional PCR. Furthermore, positive rates of H. parasuis detection using inf B-LAMP were higher (46.4%, 26/56) than the rates obtained with conventional PCR (33.9%, 19/56). inf B-LAMP specificity analysis demonstrated no cross-reactivity with any other swine pathogens. In conclusion, inf B-LAMP was more sensitive and faster and could be carried out in the absence of expensive equipment. Furthermore, the visual readout demonstrated great potential for the use of inf B-LAMP in the clinical detection of H. parasuis.