Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

AIM: To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads. METHODS: The MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, β-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosisinducing ligand, vascular endothelial growth factor andthe immunological markers interleukin-6(IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra- and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation.RESULTS: For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable(70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 ℃ and 25 ℃ after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers.CONCLUSION: MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.

Advance of Multiplex PCR in Rapid Detecting Transgenic Soybean Oil

Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

Multiplex planar microarrays for disease prognosis, diagnosis and theranosis

Advanced diagnostic methods and algorithms for immune disorders provide qualitative and quantitative multiplex measurement for pre-clinical prognostic and clinical diagnostic biomarkers specific for diseases.Choice of therapy is confirmed by modulating diagnostic efficacy of companion, theranotic drug concentrations.Assay methods identify, monitor and manage autoimmune diseases, or risk thereof, in subjects who have,or who are related to individuals with autoimmune disease. These same diagnostic protocols also integratequalitative and quantitative assay test protocol designs for responder patient assessment, risk analysis and management of disease when integrating multiplex planar microarray diagnostic tests, patient theranostic companion diagnostic methods and test panels for simultaneous assessment and management of dysimmune and inflammatory disorders, autoimmunity, allergy and cancer. Proprietary assay methods are provided to identify, monitor and manage dysimmune conditions, or risk thereof, in subjects with pathological alterations in the immune system, or who are related to individuals with these conditions. The protocols can be used for confirmatory testing of subjects who exhibit symptoms of dysimmunity, as well as subjects who are apparently healthy and do not exhibit symptoms of altered immune function. The protocols also provide for methods of determining whether a subject has, is at risk for, or is a candidate for disease therapy, guided by companion diagnosis and immunosuppressive therapy, as well as therapeutic drug monitoring and theranostic testing of disease biomarkers in response to immunoabsorption therapy. The multiplex test panels provide the components that are integral for performing the methods to recognized clinical standards.

Fiber Bragg Grating Sensor Array System Based on Digital Phase Generated Carrier Demodulation and Reference Compensation Method

A novel fiber Bragg grating （FBG） sensor array system based on digital phase generated carrier （PGC） demodulation and reference compensation method is proposed and set up. Experimental results confirm that the digital PGC demodulation can be used for wavelength-division-multiplexed FBG sensor array and the reference compensation method can reduce the environmental interference by approximately 40 dB in the frequency range from 20 Hz to 2 kHz. The minimum detectable wavelength-shift of the sensor system is 1 × 10^-3 pm/Hz^1/2.

生鲜乳中16种致病微生物多重荧光定量PCR集成检测技术的建立与初步应用

为满足生鲜乳中微生物快速通量检测的需求,建立多重荧光定量PCR集成方法,从而快速检测生鲜乳中潜在的16种致病微生物,通过设计致病性大肠杆菌、金黄色葡萄球菌、链球菌等目标菌株的特异性引物和探针,优化反应体系,验证方法的特异性、敏感性等,建立了稳定的4组多重集成荧光定量PCR反应体系,并利用人工污染的生鲜乳样品对所建立的方法和传统培养法进行了验证比较。结果显示:各对引物探针对目标菌株均能有效识别并扩增,每组体系中引物探针未发现交叉反应,对其余3组体系的12株非目标菌均未有特异性反应;组内和组间变异系数均低于3%;该方法对布鲁氏菌的最低检出限为10^(2) CFU/mL,对阪崎肠杆菌、志贺菌、沙门菌等7种致病微生物的最低检出限为10^(3) CFU/mL,对蜡样芽胞杆菌、弯曲杆菌、结核分枝杆菌等8种致病微生物的最低检出限为10^(4) CFU/mL;所建方法和传统培养法对人工污染不同致病菌的各25份样品检测结果基本一致。结果表明,本研究建立的多重集成荧光定量PCR方法灵敏度高,特异性及重复性好,可实现对生鲜乳样品中多种致病微生物的快速高效检测,为生鲜乳微生物性风险监测提供了技术支撑。

膨润土同步提纯钠化研究及在球团中应用

基于某企业生产的低品质钙基膨润土,采用多重钠化工艺进行提质改性,系统研究了多重钠化剂种类及用量对膨润土性能指标和收得指标的影响。结果表明,在钠化剂Na2CO3用量为2.0%时,钠化土的收得率为83.55%,其膨胀指数、吸水率和吸蓝量分别由原土的5.0 mL/g、119%/(2 h)和26.9 g/(100 g)提升到22 mL/g、377.1%/(2 h)和39.6 g/(100 g),达到一级钠化土指标。采用多重钠化改性工艺可以显著提升膨润土指标,钠化剂配比为2.0%Na6P6O18和2.0%Na2CO3时,双重钠化土的膨胀指数为77 mL/g,吸水率为631.8%/(2 h),吸蓝量为39.1 g/(100 g),收得率为61.29%。造球实验中添加量为1.8%时,生球落下强度由原土的3.0次/(0.5 m)分别提高至一次钠化土的4.6次/(0.5 m)和双重钠化土的8.6次/(0.5 m)。

D2D通信复用异构蜂窝网络中分布式路由资源分配方法

由于设备到设备(Device-To-Device,D2D)通信复用异构蜂窝网络环境的复杂性和动态性,难以实现高效、均衡的路由资源利用,文章提出D2D通信复用异构蜂窝网络中分布式路由资源分配方法。采用分布式系统模型构建D2D通信复用异构蜂窝网络的拓扑结构,基于邻居节点信息交换实现D2D设备发现,并引入自适应的会话建立策略建立通信链路,采用贪婪算法为D2D用户分配子信道,实现路由资源的均衡分配。实验结果表明,设计方法在提升D2D用户之间数据传输速率方面具有较好的性能,适用于D2D通信复用异构蜂窝网络中的路由资源分配。

水禽4种常见致病菌多重PCR方法的建立及应用

为建立一种能同时检测大肠杆菌、鸭疫里默氏杆菌、多杀性巴氏杆菌、金黄色葡萄球菌的多重PCR方法,试验根据大肠杆菌phoA基因、鸭疫里默氏杆菌ompA基因、多杀性巴氏杆菌kmt 1基因、金黄色葡萄球菌FemB基因的保守区域设计4对特异性引物,通过优化退火温度和引物浓度建立多重PCR方法,检验该方法的特异性、敏感性性、重复性,应用该方法检测145份病死水禽临床样品,并与传统细菌分离鉴定方法进行比较。结果表明:优化后的最佳退火温度为52℃,引物浓度为10μmol/L。建立的多重PCR方法能特异性地扩增出靶基因,沙门氏菌、链球菌、产气荚膜梭菌等临床常见菌均未扩增出条带,对大肠杆菌、鸭疫里默氏杆菌、多杀性巴氏杆菌、金黄色葡萄球菌4种细菌的检出下限是0.4 pg/mL,单一PCR扩增ompA、kmt 1、phoA和FemB基因的检出下限分别为4×10^(-3),0.4,4×10^(-3),4×10^(-6)pg/mL。多重PCR方法对临床样品的检测结果与传统细菌分离鉴定方法的符合率均在90%以上。说明试验建立的多重PCR方法具有结果稳定、重复性高的特点,适合水禽病原微生物流行病学调查。

实验动物三种条件致病菌多重分子检测方法的建立

目的建立金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌三种条件性致病菌的多重荧光定量PCR(qPCR)检测方法。方法选取金黄色葡萄球菌nuc基因、肺炎克雷伯杆菌phoE基因、绿脓假单胞菌gyrB基因设计特异性引物及探针,建立多重qPCR方法,并测试其特异性、灵敏度、标准曲线及重复性等性能指标。用该方法检测30个临床粪便样品、6个阳性参考品,检测结果与分离培养法结果进行对比,计算符合率。结果多重qPCR检测方法对金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌的标准菌株基因组DNA均能稳定检出;对鼠伤寒沙门菌、嗜肺巴斯德杆菌、鼠棒状杆菌、支气管鲍特杆菌等病原微生物项目无非特异性扩增;针对金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌标准菌株基因组DNA的灵敏度分别为1.0×10^(-5)、1.0×10^(-4)、1.0×10^(-5)ng/μL;批间差异、批内差异均小于2%,重复性良好;临床样品和阳性参考品的检测结果显示多重qPCR法与分离培养法的检测结果一致。结论本研究成功建立了一种可同时、快速检测金黄色葡萄球菌、肺炎克雷伯杆菌、绿脓假单胞菌的多重qPCR检测方法。

陈旧性骨骼DNA提取技术的研究

对陈旧性骨骼建立一个高回收率并能除去 PCR 反应抑制物的提取 DNA 的方法。采用 CTAB 法提取 DNA。结果显示,该方法不但能有效去除 PCR 抑制物,而且对水泡、火烧、土埋以及10年左右的骨骼所提取的 DNA 均能成功地进行荧光标记 STR 多基因座扩增检验。实验表明,该方法稳定,重复性好,适合陈旧骨骼标本的 DNA 提取。

上海地区长足大竹象初报

长足大竹象是慈孝竹上危害性较大的害虫 ,文章首次报道了其在上海的发生。在上海浦东局部地区 ,已有 63 2 8%的慈孝竹新笋受其危害而不能成竹。该虫在上海 1a发生 1代 ,以成虫在土中蛹室内越冬 ,7月上旬成虫开始出土 ,持续至 10月上旬 ,幼虫危害的高峰期在 7- 9月 ,7月下旬- 10月下旬化蛹 ,8月上旬 - 11月上旬成虫在土中羽化。宝发 1号、氧化乐果、乐斯本、阿克泰等 4种农药喷雾防治长足大竹象 ,校正死亡率分别为 :31 70 %、31 4 2 %、31 4 2 %及 9 0 9%。

食源性致病菌多重分子生物学检测技术研究进展

快速、可靠的食源性致病菌高通量检测方法对于确保食品安全具有重要意义,近年基于DNA水平的多重分子生物学检测技术迅速发展,针对各种不同的食源性致病菌建立了多种多重分子检测技术,包括多重PCR、多重实时荧光PCR以及基因芯片等。对这些多重分子检测技术的最新研究进展作一综述,并且建议在今后该技术的研究中,仍需要在食品中多种致病菌同时选择性增菌培养、亚致死损伤修复以及检测内标的构建等方面取得突破,从而能够更好地实现食源性致病菌的高通量检测。

多规格油套管漏磁检测方法研究

对油套管现有漏磁无损检测方案进行了对比分析,指出了各种方案的优缺点及适用范围。为适合多规格油套管的检测,提出了横向检测探靴条状化的方法。为采集大数量阵列霍尔信号,提出了1种特殊的时分复用方法,并给出了1套综合检测系统设计方案。

高密度数字全息存储方案的比较

介绍了全息存储的基本原理 ,总结了多种实现高密度海量全息存储的复用方案 ,对这些方案进行了比较与评价 .

荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立

针对肉制品中易污染的荧光假单胞菌、沙门氏菌和单增李斯特菌等有害微生物,通过3种标准菌株及肉制品建立多重聚合酶链式反应方法,实现肉制品中这3种菌的同时、快速检测。利用荧光假单胞菌的gyrB基因、沙门氏菌的invA基因和单增李斯特菌的hly A基因设计3对特异性引物,在确定引物特异性的基础上,对3种标准菌株及在冷却肉上过夜富集后进行灵敏度检测。结果表明,该多重聚合酶链式反应方法对于同时检测这3种有害微生物具有高度的特异性;同时检测这3种菌时,纯菌DNA检测限可达1 pg/μL;将3种菌一起接种到冷却肉中35℃过夜培养后,荧光假单胞菌、沙门氏菌和单增李斯特菌的检测限分别可达到9、5、70 CFU/m L。

转基因抗草甘膦油菜的实用PCR检测方法

利用定性PCR技术 ,建立了检测孟山都公司培育的转基因抗草甘膦油菜中的epsps、gox和fmv35s的方法 ,同时设立了阳性内对照napin。该方法的检测灵敏度为 0 .0 5 %。利用复合PCR可以在一个PCR反应中同时检测出epsps、gox、fmv35s和napin基因。这种方法可以有效地用于转基因抗草甘膦油菜中的外源基因的检测。

光学教学改革的探索与实践

以在基础理论教学中穿插光纤通信的密集波分复用技术为例,论述了将现代光学前沿课题与基础理论相结合,促进光学教学改革的想法和做法。

靶序列富集多重PCR-液相芯片联合检测4种常见呼吸道病毒的初步应用

目的呼吸道病毒是引起婴幼儿呼吸道感染的最主要病原体,为快速准确诊断其感染,文中建立一种基于液相芯片系统的4种常见呼吸道病毒快速分子检测方法,为有效防控其发生和流行提供有力手段。方法采集南京总医院儿科就诊的有急性呼吸道感染症状的患儿咽拭子标本120例及30例正常对照标本,分别纳入患儿组和对照组。针对A型流感病毒(Flu A)、B型流感病毒(Flu B)、呼吸道合胞病毒A型(RSVA)和B型(RSVB)的保守区序列分别设计并合成了特异性引物、探针和通用引物,构建4种病原体阳性参比品,通过对扩增和杂交条件的优化,建立了靶序列富集多重PCR(Tem-PCR)和Luminex液相芯片(x MAP)技术相结合的检测系统,并对该检测系统进行了特异性、灵敏度评价。收集以急性上呼吸道感染为主要表现的患儿咽拭子标本,用上述建立的方法进行检测分析,分别与单病毒基因荧光定量PCR检测方法及x TAG液相芯片法进行比对检测。结果建立了Flu A、Flu B、RSVA和RSVB4种呼吸道病毒的特异性快速分子检测方法且灵敏度可达10拷贝/μL。用快速分子检测方法和单病毒基因荧光定量PCR法对120例疑似患儿咽拭子标本进行检测,快速法阳性检出率为31.7%(38/120),荧光定量法阳性检出率为29.2%(35/120)。经一致性检验分析提示2种方法一致性较强(k>0.7)。部分标本同时采用x TAG液相芯片试剂盒进行检测,经一致性检验分析提示2种方法一致性较好(k>0.6)。结论临床的初步应用证实了4种常见呼吸道病毒快速分子检测方法具备灵敏、特异、快速等优点,为临床早期、准确判断呼吸道感染的病原体提供实验依据。

制图类课程“多元化”教学改革与实践

介绍了一种新型的"多元化"教学方法以及这种教学方法应用于制图类课程教学实践的情况。这种教学方法对原有制图课程体系进行"整合"、"优化",建立培养学生素质、创新设计能力的新的现代工程制图课程教学体系。在此基础上,我们运用传统教学、多媒体教学、网络教学等多种教学手段,综合各种教学手段的优点和长处,应用于制图课程教学之中。