中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2015年
1期
3-8
,共6页
李春凤%张平平%王晓英%刘晓%赵勇%孙崇云%王成彬%杨瑞馥%周蕾
李春鳳%張平平%王曉英%劉曉%趙勇%孫崇雲%王成彬%楊瑞馥%週蕾
리춘봉%장평평%왕효영%류효%조용%손숭운%왕성빈%양서복%주뢰
耶尔森菌,鼠疫%芽孢杆菌,炭疽%布鲁杆菌属%上转发光技术%免疫层析
耶爾森菌,鼠疫%芽孢桿菌,炭疽%佈魯桿菌屬%上轉髮光技術%免疫層析
야이삼균,서역%아포간균,탄저%포로간균속%상전발광기술%면역층석
Yersinia pestis%Bacillus anthracis%Brucella%Up-converting phosphor technology based lateral flow assay%Immunochromatography
目的 研制基于上转发光免疫层析技术(UPT-LF)检测鼠疫耶尔森菌(简称鼠疫菌)、炭疽杆菌芽孢、布鲁菌的试纸,与BioThreat Alert(R)免疫层析(BTA)试纸的检测和操作性能进行对比.方法 以上转发光纳米颗粒作为生物示踪物,采用双抗体夹心模式分别研制检测鼠疫菌、炭疽杆菌芽孢、布鲁菌的UPT-LF试纸.分别配制1010、109、108、107、106、105、0 CFU/ml系列浓度的鼠疫菌、炭疽杆菌芽孢、布鲁菌标准品以及109 CFU/ml系列浓度的27种特异性评价菌株菌液,采用UPT-LF试纸对其进行检测,对3种UPT-LF试纸的敏感性、精密性、线性和特异性进行分析评价.配制模拟阳性样品,分别采用UPT-LF和BTA试纸对107、106、105、0 CFU/ml系列浓度的标准品以及模拟阳性样品进行检测,比较UPT-LF和BTA试纸的检测时间、敏感性以及鼠疫菌、炭疽杆菌芽孢、布鲁菌的检出率.结果 鼠疫菌UPT-LF、炭疽杆菌芽孢UPT-LF、布鲁菌UPT-LF试纸的敏感性均达到105 CFU/ml,各系列浓度检测带信号/质控带信号(T/C)值的变异系数均≤15%,lg[T/C值-临界值(cut-off值)]与lg(标准品浓度)的相关系数分别为0.996、0.998、0.999(F=1 647.57、743.51、1 822.17,P值均<0.001).鼠疫菌UPT-LF和布鲁菌UPT-LF试纸检测特异性评价菌株时,均无非特异性交叉反应,炭疽杆菌芽孢UPT-LF试纸检测枯草芽孢杆菌分离株2#芽孢、蜡样芽孢杆菌分离株l#芽孢存在非特异交叉反应,检测其他特异性评价菌株均无非特异性交叉反应.UPT-LF试纸检测鼠疫菌、炭疽杆菌芽孢、布鲁菌样品的时间分别为33、36、37 min,BTA为115、115、111 min;UPT-LF和BTA试纸检测0 CFU/ml标准品的阴性率均为5/5;UPT-LF试纸检测炭疽杆菌芽孢、鼠疫菌、布鲁菌的敏感性均达到l05 CFU/ml,BTA试纸分别为106、106、105 CFU/ml;模拟样品检测中,UPT-LF试纸炭疽杆菌芽孢、鼠疫菌、布鲁菌的检出率均为16/16,BTA试纸分别为7/16、16/16、16/16.结论 UPT-LF试纸检测鼠疫菌、炭疽杆菌芽孢、布鲁菌的敏感性较高,特异性、线性和精密性较佳,检测和操作性能较BTA试纸更为优良.
目的 研製基于上轉髮光免疫層析技術(UPT-LF)檢測鼠疫耶爾森菌(簡稱鼠疫菌)、炭疽桿菌芽孢、佈魯菌的試紙,與BioThreat Alert(R)免疫層析(BTA)試紙的檢測和操作性能進行對比.方法 以上轉髮光納米顆粒作為生物示蹤物,採用雙抗體夾心模式分彆研製檢測鼠疫菌、炭疽桿菌芽孢、佈魯菌的UPT-LF試紙.分彆配製1010、109、108、107、106、105、0 CFU/ml繫列濃度的鼠疫菌、炭疽桿菌芽孢、佈魯菌標準品以及109 CFU/ml繫列濃度的27種特異性評價菌株菌液,採用UPT-LF試紙對其進行檢測,對3種UPT-LF試紙的敏感性、精密性、線性和特異性進行分析評價.配製模擬暘性樣品,分彆採用UPT-LF和BTA試紙對107、106、105、0 CFU/ml繫列濃度的標準品以及模擬暘性樣品進行檢測,比較UPT-LF和BTA試紙的檢測時間、敏感性以及鼠疫菌、炭疽桿菌芽孢、佈魯菌的檢齣率.結果 鼠疫菌UPT-LF、炭疽桿菌芽孢UPT-LF、佈魯菌UPT-LF試紙的敏感性均達到105 CFU/ml,各繫列濃度檢測帶信號/質控帶信號(T/C)值的變異繫數均≤15%,lg[T/C值-臨界值(cut-off值)]與lg(標準品濃度)的相關繫數分彆為0.996、0.998、0.999(F=1 647.57、743.51、1 822.17,P值均<0.001).鼠疫菌UPT-LF和佈魯菌UPT-LF試紙檢測特異性評價菌株時,均無非特異性交扠反應,炭疽桿菌芽孢UPT-LF試紙檢測枯草芽孢桿菌分離株2#芽孢、蠟樣芽孢桿菌分離株l#芽孢存在非特異交扠反應,檢測其他特異性評價菌株均無非特異性交扠反應.UPT-LF試紙檢測鼠疫菌、炭疽桿菌芽孢、佈魯菌樣品的時間分彆為33、36、37 min,BTA為115、115、111 min;UPT-LF和BTA試紙檢測0 CFU/ml標準品的陰性率均為5/5;UPT-LF試紙檢測炭疽桿菌芽孢、鼠疫菌、佈魯菌的敏感性均達到l05 CFU/ml,BTA試紙分彆為106、106、105 CFU/ml;模擬樣品檢測中,UPT-LF試紙炭疽桿菌芽孢、鼠疫菌、佈魯菌的檢齣率均為16/16,BTA試紙分彆為7/16、16/16、16/16.結論 UPT-LF試紙檢測鼠疫菌、炭疽桿菌芽孢、佈魯菌的敏感性較高,特異性、線性和精密性較佳,檢測和操作性能較BTA試紙更為優良.
목적 연제기우상전발광면역층석기술(UPT-LF)검측서역야이삼균(간칭서역균)、탄저간균아포、포로균적시지,여BioThreat Alert(R)면역층석(BTA)시지적검측화조작성능진행대비.방법 이상전발광납미과립작위생물시종물,채용쌍항체협심모식분별연제검측서역균、탄저간균아포、포로균적UPT-LF시지.분별배제1010、109、108、107、106、105、0 CFU/ml계렬농도적서역균、탄저간균아포、포로균표준품이급109 CFU/ml계렬농도적27충특이성평개균주균액,채용UPT-LF시지대기진행검측,대3충UPT-LF시지적민감성、정밀성、선성화특이성진행분석평개.배제모의양성양품,분별채용UPT-LF화BTA시지대107、106、105、0 CFU/ml계렬농도적표준품이급모의양성양품진행검측,비교UPT-LF화BTA시지적검측시간、민감성이급서역균、탄저간균아포、포로균적검출솔.결과 서역균UPT-LF、탄저간균아포UPT-LF、포로균UPT-LF시지적민감성균체도105 CFU/ml,각계렬농도검측대신호/질공대신호(T/C)치적변이계수균≤15%,lg[T/C치-림계치(cut-off치)]여lg(표준품농도)적상관계수분별위0.996、0.998、0.999(F=1 647.57、743.51、1 822.17,P치균<0.001).서역균UPT-LF화포로균UPT-LF시지검측특이성평개균주시,균무비특이성교차반응,탄저간균아포UPT-LF시지검측고초아포간균분리주2#아포、사양아포간균분리주l#아포존재비특이교차반응,검측기타특이성평개균주균무비특이성교차반응.UPT-LF시지검측서역균、탄저간균아포、포로균양품적시간분별위33、36、37 min,BTA위115、115、111 min;UPT-LF화BTA시지검측0 CFU/ml표준품적음성솔균위5/5;UPT-LF시지검측탄저간균아포、서역균、포로균적민감성균체도l05 CFU/ml,BTA시지분별위106、106、105 CFU/ml;모의양품검측중,UPT-LF시지탄저간균아포、서역균、포로균적검출솔균위16/16,BTA시지분별위7/16、16/16、16/16.결론 UPT-LF시지검측서역균、탄저간균아포、포로균적민감성교고,특이성、선성화정밀성교가,검측화조작성능교BTA시지경위우량.
Objective To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis,Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert(R) (BTA)test strips(Tetracore Inc.,USA).MethodsUsing up-converting phosphor nano-particles (UCP-NPs) as the bio-marker,three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF,Anthrax-UPT-LF,Brucella-UPT-LF were prepared and its sensitivity,accuracy,linearity and specificity were determined by detecting 1010,109,108,107,106,105 and 0 CFU/ml series of concentrations of Y.pestis,B.anthracis,Brucella standards and other 27 kinds of 109 CFU/ml series of contrations of bacteria strains.Furthermore,the speed,sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system.Results The detection limit of Plague-UPT-LF,Anthrax-UPT-LF and Brucella-LF was 105 CFU/ml.The CV of series of bacteria concentrations was ≤ 15%,and the r between lg(T/C-cut-off)and lg (concentration) was 0.996,0.998 and 0.999(F values were 1 647.57,743.51 and 1 822.17.All the P values were <0.001),respectively.The specificity of Plague-UPT-LF and Brucella-LF were excellent,while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B.subtilis and one B.cereus.Onsite evaluation showed the detection time of UPT-LF for all Y.pestis,B.anthracis spore and Brucella spp.was 33,36 and 37 min,while BTA was 115,115 and 111 min,which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA.The negative rate of two methods for blank standard was both 5/ 5,the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp.was all 105 CFU/ml,then BTA was 106,106 and 105 CFU/mnl,respectively.The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16,while BTA for B.anthracis was 7/16 only.Conclusion The good performance including rapidness,simplicity and high sensitivity will bring the bright future of UPT-LF to be broadly used on-site as first response to bio-terrorism.