基于滚环扩增技术的化学发光共振能量转移体系检测金黄色葡萄球菌
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- 英文论文题名：A chemiluminescence resonance energy transfer aptasensor based on rolling circle amplification for detection of Staphylococcus aureus
- 论文作者：郝丽玲 ; 王周平
- 英文论文作者：Hao Liling ; Wang Zhouping ; School of Food Science and Technology ; Jiangnan University
- 年：2016
- 作者机构：江南大学食品学院;
- 论文关键词：滚环扩增 ; 异鲁米诺修饰的花状纳米金 ; WS_2 ; 化学发光共振能量转移 ; 金黄色葡萄球菌
- 英文论文关键词：rolling circle amplification ; ABEI-AuNFs ; WS_2 ; chemiluminescence resonance energy transfer ; staphylococcus aureus
- 会议召开时间：2016-11-09
- 会议录名称：中国食品科学技术学会第十三届年会论文摘要集
- 英文会议录名称：Abstracts of the 13th Annual Meeting of CIFST
- 语种：中文
- 分类号：TS207.4
- 学会代码：ZGSP
- 会议名称：中国食品科学技术学会第十三届年会
- 会议地点：中国北京
- 主办单位：中国食品科学技术学会
- 学会名称：中国食品科学技术学会
- 页数：2
- 文件大小：114k
- 原文格式：O
- 会议级别：全国
摘要
本文结合滚环扩增(RCA)技术,以Co~(2+)/ABEI-AuNFs作为能量供体,以层状WS_2作为能量受体构建了化学发光共振能量转移体系实现对金黄色葡萄球菌的检测。目的:金黄色葡萄球菌是最常见的食源性致病菌之一,极少量的菌体存在就会对人体健康造成很大的威胁。因此建立快速,灵敏,高效的检测方法意义重大。方法:首先将金黄色葡萄球菌适配体与RCA引物进行DNA杂交,在靶标存在时,适配体会与靶标结合引起引物的脱落。然后,加入锁式探针,RCA反应得以启动。RCA产物互补链修饰的ABEI-AuNFs(cDNA-ABEI-AuNFS)可与RCA产物进行多位点结合。最后,向体系中加入WS_2,未结合cDNA-ABEI-AuNFs与WS_2发生化学发光共振能量转移而引起化学发光的淬灭。因此,体系的化学发光强度与靶标浓度正相关。结果与讨论:在最优的检测条件下,化学发光强度与金黄色葡萄球菌浓度的负对数线性相关,其线性范围为50cfu/mL-1.5×10~5 cfu/mL(R~2=0.9913),检测限为15cfu/mL。特异性结果结果显示该方法特异性良好。猪肉中实际样品检测与平板涂布方法进行时照,检测结果一致性良好。
We have developed a facile chemiluminescence system for the detection of Staphylococcus aureus(S.aureus),using rolling circle amplification(RCA),layered tungsten disulfide(WS_2),and Co~(2+) enhanced N?(aminobutyl)-N-(ethylisoluminol)(ABEI) functional flowerlike gold nanoparticles(AuNFs).Purpose:The Gram-positive bacterium Staphylococcus aureus(S.aureus) is one of the most common food borne pathogen for humans and animals.S.aureus can contaminate various kinds of foods.And even trace concentrations of bacterial pathogens in food can cause threats to human health.It is therefore critical to develop rapid,sensitive and selective methods to monitor 5.aureus in food.Methods:A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of S.aureus with Co~(2+) enhanced ABEI-AuNFs as donor and WS_2 nanosheet as acceptor The aptamer of S.aureus and primer of RCA was partially complemented,and in the presence of S.aureus,the aptamer tends to combine with the target thus leading to the melting of DNA duplex.When adding padlock probe that is complementary to the primer,RCA can be started.Partially complementary sequence of RCA product functional ABEIAuNFs(cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product containing multiple repetitive sequences.This cDNA-ABEI-AuNFs is less adsorbed onto the WS_2 nanosheet,thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS_2.In the absence of target S.aureus(and hence the absence of RCA and duplex formation),the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS_2 nanosheet and chemiluminescence quenching ensues.Results and Discussion:Under optimal conditions,the logarithmic correlation between the concentration of S.aureus and the CL signal was found to be linear within the range of 50cfu/mL to 1.5 × 10~5 cfu/mL(R~2= 0.9913).The limits of detection of the developed method were found to be 15cfu/mL for S.aureus.The influences of some other pathogenic bacteria including Salmonella typhimurium,Escherichia coli,Listeria monocytogenes and Bacillus cereus were used to access the selectivity of the CL biosensor,results demonstrate that the method has excellent selectivity due to the high affinity and specificity advantage of the aptamer.The method was also used to detect S.aureus in real pork samples.The results were compared with those obtained from the plate-counting methods and showed good consistency.
We have developed a facile chemiluminescence system for the detection of Staphylococcus aureus(S.aureus),using rolling circle amplification(RCA),layered tungsten disulfide(WS_2),and Co~(2+) enhanced N?(aminobutyl)-N-(ethylisoluminol)(ABEI) functional flowerlike gold nanoparticles(AuNFs).Purpose:The Gram-positive bacterium Staphylococcus aureus(S.aureus) is one of the most common food borne pathogen for humans and animals.S.aureus can contaminate various kinds of foods.And even trace concentrations of bacterial pathogens in food can cause threats to human health.It is therefore critical to develop rapid,sensitive and selective methods to monitor 5.aureus in food.Methods:A chemiluminescence resonance energy transfer aptasensor was fabricated for the detection of S.aureus with Co~(2+) enhanced ABEI-AuNFs as donor and WS_2 nanosheet as acceptor The aptamer of S.aureus and primer of RCA was partially complemented,and in the presence of S.aureus,the aptamer tends to combine with the target thus leading to the melting of DNA duplex.When adding padlock probe that is complementary to the primer,RCA can be started.Partially complementary sequence of RCA product functional ABEIAuNFs(cDNA-ABEI-AuNFs) were then annealed to multiple sites of the RCA product containing multiple repetitive sequences.This cDNA-ABEI-AuNFs is less adsorbed onto the WS_2 nanosheet,thus attenuating the quenching of ABEI-AuNFs chemiluminescence by WS_2.In the absence of target S.aureus(and hence the absence of RCA and duplex formation),the free cDNA-ABEI-AuNFs is completely adsorbed onto the WS_2 nanosheet and chemiluminescence quenching ensues.Results and Discussion:Under optimal conditions,the logarithmic correlation between the concentration of S.aureus and the CL signal was found to be linear within the range of 50cfu/mL to 1.5 × 10~5 cfu/mL(R~2= 0.9913).The limits of detection of the developed method were found to be 15cfu/mL for S.aureus.The influences of some other pathogenic bacteria including Salmonella typhimurium,Escherichia coli,Listeria monocytogenes and Bacillus cereus were used to access the selectivity of the CL biosensor,results demonstrate that the method has excellent selectivity due to the high affinity and specificity advantage of the aptamer.The method was also used to detect S.aureus in real pork samples.The results were compared with those obtained from the plate-counting methods and showed good consistency.
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