Long Fei, Zhu Xin-Na, Zhang Zhong-Ming, Shi Xian-Ming
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P.R. China.
Diagn Microbiol Infect Dis. 2008 Dec;62(4):374-81. doi: 10.1016/j.diagmicrobio.2008.08.012. Epub 2008 Oct 18.
A novel real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for rapid and accurate detection of Listeria monocytogenes. In this Q-PCR assay, a computational DNA random shuffling method was used to design an internal amplification control (IAC) sequence, which was the same in length and G + C content to the hly amplicon. This IAC sequence was inserted into the genome of L. monocytogenes to create a mutant strain named L. monocytogenes-IAC. The LM-IAC was used as an internal control during the PCR assay and produced accurate quantification of L. monocytogenes due to similar DNA extraction and amplification efficiencies between LM-IAC strain and wild-type L. monocytogenes. Quantification by this method was over a 5-log linearity range of initial L. monocytogenes with an R(2) value of 0.9997. This PCR method will provide accurate quantification of L. monocytogenes and can be used in the clinic and food assays for diagnostic purposes.
开发了一种新型实时定量聚合酶链反应（Q-PCR）检测方法，用于快速准确地检测单核细胞增生李斯特菌。在这种Q-PCR检测方法中，采用计算DNA随机改组方法设计了一个内部扩增对照（IAC）序列，其长度和G+C含量与hly扩增子相同。将该IAC序列插入单核细胞增生李斯特菌基因组中，创建了一个名为单核细胞增生李斯特菌-IAC的突变菌株。在PCR检测过程中，LM-IAC用作内部对照，由于LM-IAC菌株与野生型单核细胞增生李斯特菌之间的DNA提取和扩增效率相似，因此能够对单核细胞增生李斯特菌进行准确的定量。通过该方法进行的定量在初始单核细胞增生李斯特菌的5个对数线性范围内，R(2)值为0.9997。这种PCR方法将提供单核细胞增生李斯特菌的准确定量，可用于临床和食品检测以进行诊断。
