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.

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)。

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。

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。

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.

多重RT-PCR同时检测6种马铃薯病毒及1种类病毒

病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法,可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因,包括马铃薯Y病毒(potato virus Y,PVY)、马铃薯M病毒(potato virus M,PVM)、马铃薯S病毒(potato virus S,PVS)、马铃薯X病毒(potato virus X,PVX)、马铃薯A病毒(potato virus A,PVA)、马铃薯卷叶病毒(potato leaf curl virus,PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段,产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测,其检测灵敏度在1.6×10^(-3)~1.8×10^(-1) ng/μL。用该方法检测田间未知样品,结果与DAS-ELISA检测结果高度相符,同时PVY、PVM、PVS、PVX 4种病毒的检出率高于DAS-ELISA,表明该方法准确可靠,可用于以上马铃薯主要病毒和类病毒的快速检测。

广东省蒲瓜病毒种类鉴定及多重RT-PCR检测方法的建立

【目的】探明侵染广东省蒲瓜的病毒种类,建立一种可以同时检测多种蒲瓜病毒的RT-PCR检测方法,提高蒲瓜病毒种类鉴定效率,为蒲瓜病毒病的精准监测与防控提供依据。【方法】2018—2022年间,从广东省广州市、惠州市、清远市、汕头市和湛江市的蒲瓜主要种植区采集蒲瓜病毒病疑似样品78份,对采集的样品按照地区和症状进行分类,提取每份样品的总RNA,将一个地区具有相同症状样品的RNA等量混合后进行小RNA深度测序分析。根据小RNA深度测序分析结果,设计特异引物进行RT-PCR验证,从而明确侵染广东省蒲瓜的病毒种类。挑选6种检出率高、危害严重的蒲瓜病毒,根据GenBank数据库设计多重PCR检测引物,通过优化反应体系和反应条件,建立同时检测6种蒲瓜病毒的多重RT-PCR方法。【结果】从采集的78份蒲瓜疑似病毒病样品中,共检测到12种病毒,按照检出率从高到低分别为葫芦内源RNA病毒(Lagenaria sicerariaalphaendornavirus,LSEV)(64.1%)、黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)(62.8%)、小西葫芦虎纹花叶病毒(zucchini tigre mosaic virus,ZTMV)(51.3%)、西瓜绿斑驳花叶病毒(watermelon green mottle mosaic virus,WGMMV)(43.6%)、西瓜病毒A(watermelon virus A,WVA)(32.1%)、番木瓜环斑病毒(papaya ringspot virus,PRSV)(19.2%)、小西葫芦黄花叶病毒(zucchini yellow mosaic virus,ZYMV)(9.0%)、瓜类蚜传黄化病毒(cucurbit aphid-borne yellows virus,CABYV)(7.7%)、中国南瓜曲叶病毒(squash leaf curl China virus,SLCCNV)(7.7%)、瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)(5.1%)、瓜类黄矮失调病毒(cucurbit yellow stunting disorder virus,CYSDV)(3.8%)、甜瓜黄斑病毒(melon yellow spot virus,MYSV)(1.3%)。78份样品中,复合侵染率高达80.8%,其中2、3、4、5、6和7种病毒复合侵染的检出率分别为14.1%、16.7%、23.1%、17.9%、7.7%和1.3%。在多重RT-PCR体系中先加入WVA+CCYV+ZTMV+PRSV引物,12个反应后再加入CGMMV+WGMMV引物,引物体积依次为WVA 0.5μL、CCYV 0.5μL、CGMMV 0.4μL、WGMMV 0.4μL、ZTMV 0.6μL、PRSV 0.6μL,从而确立了一种同时扩增6种病毒的多重RT-PCR检测方法。【结论】危害广东省蒲瓜的主要病毒有12种,其中CGMMV、ZTMV、WGMMV、WVA为优势病毒;复合侵染现象较普遍,以3、4和5种病毒的复合侵染形式占比较高;研发出一种同时检测蒲瓜上6种病毒的多重RT-PCR检测技术,提高了蒲瓜病毒种类鉴定效率。

呼吸道感染患儿血浆中12种细胞因子的变化特征及其临床意义

目的:探讨12种细胞因子(IFN-α、IFN-γ、TNF-α、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10、IL-12p70和IL-17)在常见呼吸道感染(急性上呼吸道感染、支气管肺炎和急性化脓性扁桃体炎)患儿血浆中水平的变化特征及其临床意义。方法:收集2020年4月至2022年7月徐州市中心医院收治的诊断为呼吸道感染疾病的271例患儿,采用多重微球流式免疫荧光法检测外周血血浆和血清中12种细胞因子水平。结果:在呼吸道感染患儿血清中IL-6、IL-8、TNF-α和IFN-γ的水平显著高于血浆(P<0.05)。IFN-α、IFN-γ、IL-1β、IL-6、IL-8和IL-10在儿童急性上呼道感染组中的水平显著升高(P<0.05);IFN-γ、TNF-α、IL-1β、IL-6、IL-8和IL-10在支气管肺炎患儿中的水平也显著升高(P<0.05);而在急性化脓性扁桃体炎患儿中12种细胞因子水平变化差异无统计学意义(P>0.05)。结论:儿童常见呼吸道感染发病过程中外周血血浆和血清中特定细胞因子谱会发生改变,可作为判断上述疾病病情严重程度的临床免疫学监测指标。

一种基于3×3光纤耦合器的短距离光纤频率传递的噪声抑制方法

提出了一种噪声抑制方法,设计了基于3×3光纤耦合器迈克尔逊干涉仪的频率传递系统,使用嵌入式系统进行控制,通过调整光纤长度,实时补偿由温度变化等环境因素引起的时延变化,并进行了实验验证。启用时延补偿后,实验用的30 m长传输光纤在环境温度变化21℃条件下长度变化量小于±1μm,对应时间延迟变化量小于10 fs,所传输的光梳重频信号的频率稳定度没有明显变化。本文工作有望为空间条件下的光钟信号向比对设备的传输路径噪声抑制提供有效的解决方法。

基于椭球法的携能通信OFDM系统能效优化算法

随着无线通信技术的快速发展,无线接入设备日益增多,但系统能耗也在不断增长。具备无线携能通信能力的正交频分复用(Orthogonal frequency division multiplexing,OFDM)系统可以有效提高系统能量效率。本文针对以系统能效为优化目标的资源分配问题,提出了基于椭球法的携能通信OFDM系统能效优化算法。该算法采用椭球法对拉格朗日乘子进行更新,可以有效加快算法收敛速度,提升算法性能。仿真实验结果表明,所提出基于椭球法的能效优化算法能有效解决以系统能效为优化目标的资源分配问题,与次梯度法相比,椭球法的收敛速度更快,能够显著地降低算法复杂度。

“两性一度”范式下沉积岩石学课程混合式教学实践——关于参加第二届黑龙江省高校教师教学创新大赛的思考

沉积岩石学课程体系庞大、内容繁多,而且其突出实践、注重应用的课程特征进一步加大了“教”与“学”难度。此外,以往教学中“课上强、课外弱”“重教材、轻需求、少思想”“方法、考核与学习资源千人一面”“理论、实践、实习三套班子”等痛点问题严重影响了教学效果。针对上述问题,以“高阶性、创新性、挑战度”作为教学设计的顶层架构并进行课程体系重构,充分结合基于MOOC的翻转课堂、沉浸式虚拟案例、情景辩论等信息化教学模式,同时配以师-生共建的多元化评价标准与多维度、常态化的学习资源开展在沉积岩石学教学中的应用实践。结果证实本次教学实践在一定程度上解决了沉积岩石学教学中的痛点问题,同时也是青年教师服务课程建设的新探索。

一种分时复用的绝对式纳米时栅位移传感器设计

现代高端装备制造对精密位移检测技术提出了绝对位置测量、高精度和大量程的需求,针对绝对式位移量程与编码的矛盾,提出了一种基于分时复用方法的绝对式纳米时栅位移传感器。采用单列式时栅位移传感器作为绝对式传感器的精测部分,记为传感器A。为了实现绝对位移测量,增加一个周期数比传感器A少且互为质数的对射增量式时栅位移传感器,记为传感器B。利用传感器A与传感器B输出行波的相位值,通过查找表和定位计算实现绝对位移测量。提出了一种分时复用的方法,通过分时间段为传感器A与传感器B提供激励信号,可以有效降低传感器功耗。此外,传感器A与传感器B的感应电极在内部用引线连接,共用1组感应信号处理接口,减少了1组感应信号处理器件,节约了电路空间。采用多层PCB工艺制造了传感样机,搭建了实验平台并对样机性能进行测试。最终实验结果表明:在450 mm范围内,非线性测量误差达到±3μm。

改进的OFDM雷达通信一体化信号峰均比抑制方法

正交频分复用(orthogonal frequency division multiplexing,OFDM)信号是雷达通信一体化系统常用的发射波形之一,但是其峰均比较高,影响发射机的工作效率。传统的限幅法虽然可以降低系统OFDM信号的峰均比(peak-to-average power ratio,PAPR),但会造成误码率(bit error rate,BER)增大和带外频谱泄露的问题。对传统的限幅法进行改进,将高于门限的信号进行抑制,通过迭代滤波消除带外信号弥散造成的频谱效率下降的问题。将改进峰均比抑制方法与传统峰均比抑制方法进行比较,验证了所提方法的低峰均比性能、误码率性能和模糊函数性能。仿真实验表明,所提方法通过合理设置限幅门限和选择迭代滤波次数,可以有效降低OFDM信号的PAPR,并且对雷达探测性能和通信性能影响较小。

基于二维光子晶体的四通道滤波器设计

为实现光电信息器件的高集成度和高效输出,提高光电信息处理能力,设计了一种基于二维光子晶体的四通道滤波器,根据4个点缺陷微腔与线缺陷波导耦合原理,引入反射异质结并调整微腔与反射结的距离,提高输出效率,对滤波器1403、1426、1449、1508 nm四波长的波分复用功能进行仿真。仿真结果表明:该器件可实现高效传输,四波长透射率均超过95%,插入损耗均小于0.23 dB,通道间串扰均小于-8.7 dB。

多重实时荧光PCR TaqMan-MGB杂交探针标记法快速鉴定转基因玉米MIR162

为对转基因玉米MIR162的进口实行监测、规范转基因玉米在国内市场中的流通,建立一种转基因玉米MIR162准确快速的检测方法,通过设计转基因玉米MIR162品系特异性序列的引物和探针,摸索和优化多重荧光PCR体系和参数,开发建立多重实时荧光PCR TaqMan-MGB杂交探针标记法快速鉴定转基因玉米MIR162。结果表明,转基因玉米MIR162品系标准品和平行样品中内标基因和品系特异性基因均出现明显扩增曲线,其他转基因玉米品系标准品仅内标基因有扩增曲线,空白对照无扩增曲线,说明该TaqMan-MGB探针对转基因玉米MIR162品系具有扩增特异性。该方法可作为鉴定筛查转基因玉米MIR162品系及其转化体成分的有效方法,用于规范管理转基因产品在市场上的流通。

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。