安徽农业科学
安徽農業科學
안휘농업과학
JOURNAL OF ANHUI AGRICULTURAL SCIENCES
2010年
2期
602-605
,共4页
郝玉芹%孙皆宜%李艾%杨国兴%张伟
郝玉芹%孫皆宜%李艾%楊國興%張偉
학옥근%손개의%리애%양국흥%장위
多重PCR%食源性致病菌%检测%FTA滤膜
多重PCR%食源性緻病菌%檢測%FTA濾膜
다중PCR%식원성치병균%검측%FTA려막
Multiplex PCR%Food-borne pathogens%Detection%FTA filter%Orthogonal experimental design
[目的]建立金黄色葡萄球菌、沙门氏菌、志贺氏菌的快速、敏感、特异的多重PCR检测方法.[方法]根据金黄色葡萄球菌的nuc基因、沙门氏菌的IpaB基因、志贺氏菌的ipaH基因,设计3对特异性引物进行多重PCR检测,利用正交设计对多重PCR反应体系的镁离子、Taq酶、dNTP、引物的浓度在4个水平上进行L_(16)(4~4)优化试验,并确定该检测方法的特异性与灵敏度.[结果]3对引物能特异性扩增出210、280、393 bp的目的条带.利用FTA滤膜提取模板DNA,采用建立多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度,灵敏度金黄色葡萄球菌为3.5×10~2cfu/ml、沙门氏菌为2.2×10~2cfu/ml、志贺氏菌为1.0×10~2cfu/ml.[结论] 该检测方法准确、快速、高效,为同时检测食品中多种致病菌奠定了基础.
[目的]建立金黃色葡萄毬菌、沙門氏菌、誌賀氏菌的快速、敏感、特異的多重PCR檢測方法.[方法]根據金黃色葡萄毬菌的nuc基因、沙門氏菌的IpaB基因、誌賀氏菌的ipaH基因,設計3對特異性引物進行多重PCR檢測,利用正交設計對多重PCR反應體繫的鎂離子、Taq酶、dNTP、引物的濃度在4箇水平上進行L_(16)(4~4)優化試驗,併確定該檢測方法的特異性與靈敏度.[結果]3對引物能特異性擴增齣210、280、393 bp的目的條帶.利用FTA濾膜提取模闆DNA,採用建立多重PCR同時對3種食源性緻病菌進行檢測具有較高的靈敏度,靈敏度金黃色葡萄毬菌為3.5×10~2cfu/ml、沙門氏菌為2.2×10~2cfu/ml、誌賀氏菌為1.0×10~2cfu/ml.[結論] 該檢測方法準確、快速、高效,為同時檢測食品中多種緻病菌奠定瞭基礎.
[목적]건립금황색포도구균、사문씨균、지하씨균적쾌속、민감、특이적다중PCR검측방법.[방법]근거금황색포도구균적nuc기인、사문씨균적IpaB기인、지하씨균적ipaH기인,설계3대특이성인물진행다중PCR검측,이용정교설계대다중PCR반응체계적미리자、Taq매、dNTP、인물적농도재4개수평상진행L_(16)(4~4)우화시험,병학정해검측방법적특이성여령민도.[결과]3대인물능특이성확증출210、280、393 bp적목적조대.이용FTA려막제취모판DNA,채용건립다중PCR동시대3충식원성치병균진행검측구유교고적령민도,령민도금황색포도구균위3.5×10~2cfu/ml、사문씨균위2.2×10~2cfu/ml、지하씨균위1.0×10~2cfu/ml.[결론] 해검측방법준학、쾌속、고효,위동시검측식품중다충치병균전정료기출.
[Objective] The research aimed to establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection of Staphylococcus aureus, Salmonella spp., and Shigella spp.[Method] Three pairs of specific primers were designed according to Staphylococcus aureus nuc gene, almonella spp.IpaB gene, higella spp.ipaH gene.Orthogonal experimental design was used to optimize multiple PCR amplification system for food-borne bacterial pathogens of four factors (Taq DNA polymerase, Mg~(2+), dNTP and primers) at four levels.The specificity and the sensitivity of this detection method were confirmed.[Result] Three DNA fragments of 210, 280 and 393 bp were amplified.Template was prepared using FTA filter and three food-borne bacterial pathogens were simultaneously detected by the established multiplex PCR technology.The sensitivity of this method was 3.5×10~2 cfu/ml for Staphylococcus aureus, 2.2×10~2 cfu/ml for Salmonella spp., and 1.0×10~2cfu/ml for Shigella spp..[Conclusion] This method was accurate, rapid and efficient in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.