CAJ | 학술논문

目的建立能够对类鼻疽伯克霍尔德菌(类鼻疽菌)进行现场快速定量检测的上转发光免疫层析(Bps-UPT-LF)方法.方法以上转发光材料(UCP)作为标记物,采用双抗体夹心法制备用于类鼻疽菌检测的Bps-UPT-LF试纸.采用分别由类鼻疽菌及与其近缘或传播途径类似的菌株配制的系列浓度标准菌悬液,评价Bps-UPT-LF试纸条的敏感度、准确度、精密度及特异度等检测性能后,采用纯生物化学试剂和模拟样本分别对其进行单因素干扰因子和实际样品的耐受性评价,通过调整该方法的液体量取体积评价其对操作误差的耐受性.结果整个检测流程20 min内可完成,Bps-UPT-LF试纸的敏感度达104 CFU/ml,定量范围为104～ 107 CFU/ml,跨越了4个数量级.浓度为107和108 CFU/ml高浓度的类鼻疽菌近缘或传播途径近似菌株对检测特异度均无影响.Bps-UPT-LF试纸除能在高浓度的酸碱物质(pH 1～12)、盐(≤2 mol/L的NaCl和KCl的混合物)、黏性物质(≤50 g/L的聚乙二醇20000、≤20％的甘油)、生物基质(≥400 g/L的牛血清白蛋白、≥80 g/L的干酪素)等单因素样本的干扰下保证检测特异度及敏感度外,还能对浓度≥400 g/L的奶粉、面粉、果珍、新鲜和腐败脏器以及浓度≤200 g/L的腻子粉、蔗糖、味精和土壤等各种动物、环境、粉末之类的实际样本进行直接检测.-50％～ 200％的样品体积偏差、-22％～ 44％样品处理液体积偏差、-30％～30％的上样混合物体积偏差等操作误差对检测特异度和敏感度无影响.结论本研究建立的基于Bps-UPT-LF技术检测类鼻疽菌的方法从检测性能、耐受性等方面均可满足自然疫源地监测和生物反恐对类鼻疽菌进行现场快速定量检测的需求.
목적 건립능구대류비저백극곽이덕균(류비저균)진행현장쾌속정량검측적상전발광면역층석(Bps-UPT-LF)방법.방법 이상전발광재료(UCP)작위표기물,채용쌍항체협심법제비용우류비저균검측적Bps-UPT-LF시지.채용분별유류비저균급여기근연혹전파도경유사적균주배제적계렬농도표준균현액,평개Bps-UPT-LF시지조적민감도、준학도、정밀도급특이도등검측성능후,채용순생물화학시제화모의양본분별대기진행단인소간우인자화실제양품적내수성평개,통과조정해방법적액체량취체적평개기대조작오차적내수성.결과 정개검측류정20 min내가완성,Bps-UPT-LF시지적민감도체104 CFU/ml,정량범위위104～ 107 CFU/ml,과월료4개수량급.농도위107화108 CFU/ml고농도적류비저균근연혹전파도경근사균주대검측특이도균무영향.Bps-UPT-LF시지제능재고농도적산감물질(pH 1～12)、염(≤2 mol/L적NaCl화KCl적혼합물)、점성물질(≤50 g/L적취을이순20000、≤20％적감유)、생물기질(≥400 g/L적우혈청백단백、≥80 g/L적간락소)등단인소양본적간우하보증검측특이도급민감도외,환능대농도≥400 g/L적내분、면분、과진、신선화부패장기이급농도≤200 g/L적니자분、자당、미정화토양등각충동물、배경、분말지류적실제양본진행직접검측.-50％～ 200％적양품체적편차、-22％～ 44％양품처리액체적편차、-30％～30％적상양혼합물체적편차등조작오차대검측특이도화민감도무영향.결론 본연구건립적기우Bps-UPT-LF기술검측류비저균적방법종검측성능、내수성등방면균가만족자연역원지감측화생물반공대류비저균진행현장쾌속정량검측적수구.
Objective To develop an up-converting phosphor technology-based lateral-flow (UPT-LF) assay for rapid quantitative detection of Burkholderia pseudomallei on site.Methods The strip Bps-UPT-LF strip was prepared with up-converting phosphor (UCP) particles as the bio-label using double-antibody sandwich method.Detection performance,including sensitivity,quantitative accuracy,precision,and specificity,were first evaluated using bacterial suspensions of Burkholderia pseudomallei,the related species and the strains which had similar routes of transmission with scrial standard concentrations diluted by phosphate buffer,then biological and chemical reagents and simulated samples with series concentrations were employed for sample tolerance evaluation,while the operation error during on site detection was also evaluated through adjusting liquid measure.Results The whole detection was accomplished within 20 minutes,and the sensitivity was 104 CFU/ml with linear quantitative range from 104 CFU/ml to 107 CFU/ml,which covered four orders of magnitude.Bps-UPT-LF strip demonstrated high specificity with the absence of any false-positive result even at 107 and 108 CFU/ml of non-specific bacterial contamination.Not only Bps-UPT-LF strip could tolerate to high concentration of the extreme acid and basic matter (pH 1-12),saline matter (≤ 2 mol/L mixture of NaCl and KCl),viscous materials (≤ 50 g/L of PEG 20000 and ≤20％ of glycerol) and bio-macromolecule (≥400 g/L of bovine serum albumin or ≥ 80 g/L of casein),but also it can directly detect animal,environmental and powder specimen,such as ≥ 400 g/L of milk powder,flour powder,fruit juice,fresh and decomposed viscera,and ≤200 g/L of putty powder,sucrose,gourmet powder,and soil.Operation errors of liquid measure had few effects on sensitivity and specificity,including-50％-200％ of sample,-22％-44％ of sample-treating buffer and -30％-30％ of loading mixture.Conclusion The good detection performance and tolerance performance bring the bright future for Bps-UPT-LF strip to detect Burkholderia pseudomallei on site rapidly and quantitatively for nature foci surveillance and anti-bioterrorism.