Abstract

Haemophilus parasuis is the causative agent of Glässer’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 diagnosingH. 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°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^-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. 

Key words: Glässer’s disease, Haemophilus parasuis, inf B, PCR, LAMP

Abbreviation

LAMP, Loop-mediated isothermal amplification method; PCR, polymerase chain reaction; TSA, tryptic soy agar; EDTA, ethylene diamine tetraacetic acid; NAD, nicotinamide adenine dinucleotide