CAJ | 학술논문

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(Listeriamonocytogenes)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EcoR I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(105～101copies/μl,R2≥0.998),灵敏度为10 copies/PCR,同步检测人工污染脱脂灭菌乳的灵敏度为102CFU/ml.
목적 건립일충쾌속、특이、령민、준학정량적단핵세포증생(단증)리사특균(Listeriamonocytogenes)여지하균(Shigella)동보검측방법.방법 분별근거단증리사특균용혈소O기인hly여지하균침습성질립항원H기인ipaH설계합성인물화탐침.구건중조질립pGEM-T-hly여pGEM-T-ipaH,병이EcoR I단매절사배상중조질립선성화작위표준품.우화반응체계,분석특이성.쌍중형광정량PCR대인공오염적탈지멸균유진행검측.결과 성공구건료중조질립표준품,병운용5'、3'단분별표기FAM、TAMRA적hly기인탐침화5'、3'단분별표기HEX、TAMRA적ipaH기인탐침성공건립료단증리사특균여지하균동보형광정량PCR검측방법.결론 건립적방법유교강적특이성,선성범위호(105～101copies/μl,R2≥0.998),령민도위10 copies/PCR,동보검측인공오염탈지멸균유적령민도위102CFU/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 monocytogencs 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 negative 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(rAM and TAMRA double labelled)and ipaH probe (HEX and TAMRA double labelled)were used to develop an optimized PCR successfuliv.Conclusion The selected primers and probes showed high specificity for these two target bacteria,the linear range of the assay was good(105-101 copies/μl,R2≥0.998)and sensitivity Was 10 copies/PCR.Following a DNA extraction method which combined EZ Spin Colum Genomic DNA Isolation Kit(BBI)/Phenol-chloroform,the sensitivity of assay Was 102CFU/ml for both Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk,which equivalents to 10 CFU/PCR.