PCR-焦磷酸测序检测单增李斯特氏菌研究

摘要 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( Listeria monocytogenes, LMO)的方法。方法：根据LMO的hly基因设计扩增引物和测序引物，特异地扩增目的片段，再制备单链模版在测序引物引导下进行焦磷酸测序，测序结果与GenBank中的hly基因序列比对判断LMO。结果：扩增引物和测序引物表现出良好的特异性，16株LMO均扩增出大小249bp的DNA片段，焦磷酸测序结果与hly基因序列100%匹配，而非LMO对照菌株未扩增出DNA条带，焦磷酸测序结果阴性。结论：建立的方法特异性高，是快速从DNA序列水平上检测LMO的有效手段。

PCR-Pyrosequencing Detection of Listeria monocytogenes

Abstract: Objective:To establish a method detecting 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,under the guidance of the sequencing primer ,pyrosequencing was used to sequence DNA base sequences. The strains were judged for LMO by aligning the sequencing results to the hly gene sequence in GenBank. Results :PCR primers and sequencing primers were demonstrated good specificity.The results showed that, 16 LMO strains could be amplified 249bp DNA fragments and the pyrosequencing results matched hly gene sequence in 100%,while both of the results of the other control strains were showed negative. Conclusion: This new established method is highly accurate and an effective means for rapid detection of LMO.