鸭源鸡杆菌双重PCR检测方法的初步建立

Preliminary development of a duplex PCR assay for detecting Gallibacterium anatis

为建立鸭源鸡杆菌(G.anatis)双重PCR检测方法,并对鸡杆菌分离株进行种类鉴定,本研究根据GenBank中G.anatis 12656-12株的gtxA基因序列设计一对PCR引物,合成扩增rpoB基因的引物作为内参基因,建立了G.anatis双重PCR检测方法。检测结果显示:以G.anatis Yu-PDS-RZ-1-SLG株DNA为模板进行的双重PCR检测能够扩增出两条目的片段;其它16株细菌包括副鸡禽杆菌、多杀性巴氏杆菌、大肠杆菌、沙门氏菌、福氏志贺菌和奇异变形杆菌均只扩增出了一条目的片段;该方法可以检测到浓度为6.5×102cfu/mL的G.anatis;34株鸡杆菌分离株均扩增出了两条预期大小的目的片段。此外,序列分析结果表明,10株鸡杆菌河南分离株的rpoB基因序列与鸭源鸡杆菌种参考株(CCUG 15563)的同源性最高(97.5%～99.0%),属于鸭源鸡杆菌种。本研究建立的G.anatis双重PCR检测方法,可用于含gtxA基因鸡杆菌菌株的鉴定及临床病原学诊断。

A duplex PCR assay was developed based on the published sequence of the gtxA gene from Gallibacterium anatis and Christensen＇s reference strain. The specificity test showed that two expected DNA fragments were amplified in the DNA sample from G.anatis Yu-PDS-RZ-1-SLG strain, whereas just one fragment was detected when using DNA templates from 16 non-Gallibacterium strains （Avibacterium paragallinarum, Pasteurella multocida, Escherichia cull, Salmonella Spp. and Proteus mirabilis）; the detection limitation of the duplex PCR were 6.5~102 cfu/mL based on the amplification of a series G.anatis culture. The positive results were also observed in the amplification of the 34 Gallibacterium isolates, furthermore, the highest homology （97.5% to 99.0%） was noticed between the 10 Gallibacterium isolates and the G.anatis reference strain （CCUG 15563）. Thus, ten Gallibacterium isolates were classified in G.anatis species, the duplex PCR assay developed in the present study is useful for identification, clinical and etiological diagnosis of gtxA-containing Gallibacterium isolates.