基于双启动引物特异性检测单核细胞增生李斯特氏菌的PCR方法

以单核细胞增生李斯特氏菌iap基因为靶基因,利用一新型PCR引物设计方法--双启动引物（Dual-priming oligonucleotide,DPO）,建立了特异性检测单核细胞增生李斯特氏菌的DPO-PCR方法,测试了DPO-PCR方法退火温度不敏感性、特异性及灵敏度,并在实践检测中进行了初步应用。结果显示:该方法检测单核细胞增生李斯特氏菌的灵敏度为1.51×102CFU/mL;退火温度不敏感性测试中,与常规PCR引物相比,DPO引物在48⁶8℃退火温度范围内均能够高效率地扩增靶基因;特异性测试中,DPO-PCR方法能特异地检测出目标菌,与其他菌株无非特异性扩增反应,比常规PCR方法显示出更强的特异性。实践应用证明,利用DPO-PCR方法对130份样本进行检测,共计检出9份单核细胞增生李斯特氏菌阳性样本,经国标法（GB/T 4789.30-2008）复检,两者检测结果一致,显示出良好的实用性,为单核细胞增生李斯特氏菌的快速准确检测提供了新方法。 

Dual-priming primers-based PCR method for specific detection of Listeria monocytogenes

In this study, a Dual-priming oligonucleotide (DPO) -based PCR method for specific detection of L.monocytogenes was developed with iap gene as target. Annealing temperature insensitivity, specificity and detection sensitivity of the DPO-PCR method were tested and its preliminary application was carried out in practice. Results showed that the detection sensitivity of the DPO-PCR method was 151CFU / mL. In the annealing temperature insensitivity test, compared with conventional PCR primers, DPO primers were able to efficiently amplify target gene in the annealing temperature range of 48⁶8℃. In the specificity test, the DPOPCR method showed a higher specificity for the target bacteria than conventional PCR method and no nonspecific amplification reactions were observed in DPO-PCR. In practice, 9 L.monocytogenes positive samples from 130 samples were detected by the DPO-PCR method, which was in accordance with the testing results according to GB/T 4789.30-2008, showing a better practicability. The DPO-PCR provided a new method for fast and accurate detection of L.monocytogenes.