基于上转发光免疫层析技术快速定量检测类鼻疽伯克霍尔德菌方法的建立

Development and systematical evaluation of an up-converting phosphor technology based lateral flow assay for quantitative detection of Burkholderia pseudomallei

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20min内可完成，Bps-UPT—LF试纸的敏感度达10^4CFU／ml，定量范围为10^4～10^7CFU／ml，跨越了4个数量级。浓度为10^7和10^8CFU／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（≤2mol／L的NaCl和KCl的混合物）、黏性物质（≤50g／L的聚乙二醇20000、≤20％的甘油）、生物基质（≥400g／L的牛血清白蛋白、≥80g/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≥400g／L的奶粉、面粉、果珍、新鲜和腐败脏器以及浓度≤200g／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。

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 serial 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 10^4 CFU/ml with linear quantitative range from 10^4 CFU/ml to 10^7 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 10^7 and 10^8 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 （ 450 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 pseudomaUei on site rapidly and quantitatively for nature loci surveillance and anti-bioterrorism.