CAJ | 학술논문

目的：探讨基因芯片技术和多重PCR技术用于检测和筛查食源性致病菌的可行性。方法通过设计靶细胞引物序列，采用生物素标记反向引物5′端，氨基基团标记寡核苷酸探针5′端。将探针在固相载体上点样，制备基因芯片，PCR产物与芯片点制探针区域进行杂交，并对PCR杂交反应的体系进行优化。结果基因芯片技术可以同时检测志贺氏菌、沙门氏菌、肺炎克雷伯菌、布鲁氏菌、奇异变形菌、金黄色葡萄球菌和空肠弯曲菌等多种病原菌，操作简便，特异性强。细菌纯培养物灵敏度为5．0×102CFU／mL，DNA检测灵敏度为0．1pg，检测分离菌株符合率为100％。利用引物建立和优化了PCR检测体系，分别确定了Mg2＋浓度和退火温度Tm值为1．5mmol／L和56℃，检测灵敏度达到10pg，此灵敏度下可以扩增出全部特异性引物条带。结论通过基因芯片技术和多重PCR技术可以有效检测食源性致病菌，为高通量筛查检测病原菌提供了新思路，值得在食品安全领域推广应用。
목적：탐토기인심편기술화다중PCR기술용우검측화사사식원성치병균적가행성。방법통과설계파세포인물서렬，채용생물소표기반향인물5′단，안기기단표기과핵감산탐침5′단。장탐침재고상재체상점양，제비기인심편，PCR산물여심편점제탐침구역진행잡교，병대PCR잡교반응적체계진행우화。결과기인심편기술가이동시검측지하씨균、사문씨균、폐염극뢰백균、포로씨균、기이변형균、금황색포도구균화공장만곡균등다충병원균，조작간편，특이성강。세균순배양물령민도위5．0×102CFU／mL，DNA검측령민도위0．1pg，검측분리균주부합솔위100％。이용인물건립화우화료PCR검측체계，분별학정료Mg2＋농도화퇴화온도Tm치위1．5mmol／L화56℃，검측령민도체도10pg，차령민도하가이확증출전부특이성인물조대。결론통과기인심편기술화다중PCR기술가이유효검측식원성치병균，위고통량사사검측병원균제공료신사로，치득재식품안전영역추엄응용。
Objective To investigate the feasibility of the gene chip technique and multiplex PCR technique for detecting and screening foodborne pathogens .Methods The primer sequences were designed to target cells ,the biotin was adopted to label the re‐verse primer 5′end and the amino group was adopted to label oligonucleotide probe 5′end .The probe spotted on a solid support for preparing microarray ,PCR product was hybridized with microarray probe region ,PCR and hybridization reaction system was opti‐mized .Results The microarray technique could simultaneously detect multiple pathogens of Shigella ,Salmonella ,Klebsiella pneu‐moniae ,Brucella ,Proteus mirabilis ,Staphylococcus aureus ,Campylobacter jejuni ,etc .,which was easy to operate and had strong specificity .The sensitivity of bacterial pure cultures was 5 .0 × 102 CFU/mL ,the sensitivity of DNA detection was 0 .1 pg ,the coin‐cidence rate for detecting isolated bacteria was 100 % .The PCR detection system was established and optimized by using primers , the concentration of Mg2+ and the annealing temperature Tm value of 1 .5 mmol/L and 56 ℃ were determined ,the detection sensi‐tivity reached to 10 pg ,all the specific primers amplified bands could be amplified under this sensitivity .Conclusion The gene chip technique and multiplex PCR technique can effectively detect foodborne pathogens ,which provide a new idea for detecting pathogens with the high‐throughput screening and are worth popularization and application in the field of food safety .