Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

Jian-min Zhang; Hai-yan Shen; Ming Liao; Tao Ren; Li-li Guo; Cheng-gang Xu; Sai-xiang Feng; Hui-ying Fan; Jing-yi Li; Ji-dang Chen; Bin Zhang

Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

2012

Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by ﬁbrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal ampliﬁcation (LAMP) test was developed to improve the speciﬁcity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly ampliﬁed the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classiﬁed into 9 serovars and had 37 genetic patterns when analysed by pulsed-ﬁeld gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, speciﬁcity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer’s disease.

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