单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立

The development of duplex real-time PCR for detection of Listeria monocytogenes and Shigella

目的建立一种快速、特异、灵敏、准确定量的单核细胞增生（单增）李斯特菌（Listeria monocytogenes）与志贺菌（Shigella）同步检测方法。方法分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针。构建重组质粒pGEM—T—hly与pGEM—T—ipaH，并以EcoRⅠ单酶切使环状重组质粒线性化作为标准品。优化反应体系，分析特异性。双重荧光定量PCR对人工污染的脱脂灭菌乳进行检测。结果成功构建了重组质粒标准品，并运用5’、3’端分别标记FAM、TAMRA的hly基因探针和5’、3’端分别标记HEX、TAMRA的ipaH基因探针成功建立了单增李斯特菌与志贺菌同步荧光定量PCR检测方法。结论建立的方法有较强的特异性，线性范围好（10^5～10^1 copies／ul，R^2≥0．998），灵敏度为10copies／PCR，同步检测人工污染脱脂灭菌乳的灵敏度为10^2CFU／ml。

Objective To develop a rapid, sensitive, specific and accurate quantitative duplex real-time PCR assay for detection of Listeria monocytogenes and Shigella. Methods Two sets of specific primers and probes were selected according to Listeria monocytogenes hly gene and Shigella ipaH gene. The target hly and ipaH fragments were amplified by PCR, and used to construct recombinant pGEM-T-hly and pGEM-T-ipaH respectively. The two recombinant circular plasmid DNAs were linearized with EcoR Ⅰ that did not cut within the target DNA fragment. The ten-fold dilutions of plasmid were subjected to the standard quantitation curve in duplex real-time PCR assay. Various genomic DNAs of Listeria innocua, Listeria weshimeri, Salmonella, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Proteus were used as nega- tive controls to confirm the specificity of duplex real-time PCR assay. The assay was also used to detect Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk. Results The recombinant plasmids were constructed successfully, hly probe （FAM and TAMRA double labelled） and ipaH probe （ HEX and TAMRA double labelled ） were used to develop an optimized PCR successfully . Conclusion The selected primers and probes showed high specificity for these two target bacteria, the linear range of the assay was good （ 10^5-10^1 copies/pal, R^2 ≥0. 998） and sensitivity was 10 copies/PCR. Following a DNA extraction method which combined EZ Spin Column Genomic DNA Isolation Kit（ BBI）/Phenol-chloroform, the sensitivity of assay was 102 CFU/ml for both Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk, which equivalents to 10 CFU/PCR.