PCR-焦磷酸测序检测单核细胞增生李斯特氏菌研究

Detection of Listeria monocytogenes by PCR-pyrosequencing

目的建立结合PCR-焦磷酸测序检测单核细胞增生李斯特氏菌的方法。方法根据单核细胞增生李斯特氏菌的hly基因设计扩增引物和测序引物,特异地扩增目的片段,再制备单链模板,在测序引物引导下进行焦磷酸测序,通过测序结果与GenBank中的hly基因序列的比对进行鉴定。结果扩增引物和测序引物表现出良好的特异性,16株单核细胞增生李斯特氏菌菌株均扩增出大小249 bp的DNA片段,焦磷酸测序结果与hly基因序列100%匹配,而阴性对照菌株均未扩增出DNA条带,焦磷酸测序结果为阴性。结论建立的方法特异性高,是快速从DNA序列水平上检测单核细胞增生李斯特氏菌的有效手段。

Objective To establish a method tO detect Listeria monocytogenes （LMO） by PCR-pyrosequencing. Methods A pair of PCR primers and a sequencing primer were designed according to the hly gene of LMO. The target gene was amplified by PCR specifically and single-stranded DNA templates for pyrosequencing were prepared from the PCR products. Finally, pyrosequencing was performed under the guidance of the sequencing primer. The strains were identified by aligning the sequencing results to the hly gene sequence in GenBank. Results PCR primers and sequencing primers showed good specificity. The results showed that a 249 bp DNA fragment was amplified from 16 LMO strains and the pyrosequencing results perfectly matched the hly gene sequence, while the control strains were both negative. Conclusion This new established method is accurate and effective for rapid detection of LMO.