Original Research

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
Onderstepoort Journal of Veterinary Research | Vol 79, No 1 | a383 | DOI: https://doi.org/10.4102/ojvr.v79i1.383 | © 2012 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 | This work is licensed under CC Attribution 4.0
Submitted: 05 October 2011 | Published: 24 April 2012

About the author(s)

Jian-min Zhang, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Hai-yan Shen, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Ming Liao, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Tao Ren, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Li-li Guo, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Cheng-gang Xu, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Sai-xiang Feng, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Hui-ying Fan, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Jing-yi Li, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Ji-dang Chen, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China
Bin Zhang, The Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, China

Abstract

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.

Keywords

Haemophilus parasuis; loop-mediated isothermal amplification; polymerase chain reaction; pulsed-field gel electrophoresis; serotyping

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Crossref Citations

1. Development of a recombinase polymerase amplification assay for rapid detection of Haemophilus parasuis in tissue samples
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