HBV DNA超敏定量检测试剂盒的性能验证

目的:验证天隆HBV DNA超敏定量检测试剂盒在cobas~?Z480全自动荧光定量PCR分析仪上的检测性能,以评估其是否满足临床应用要求。方法:参考CNAS-GL039《分子诊断检验程序性能验证指南》和CNAS-CL02-A009《医学实验室质量和能力认可准则在分子诊断领域的应用说明》等相关文件,应用临床血清样本和HBV DNA国家标准物质以及WHO国际标准物质,对精密度、正确度、线性范围、定量限、检出限、抗干扰能力、交叉污染、试剂批间差异、核酸室温放置时间等性能进行验证。结果:该系统检测10~2、10~3、10~6 IU/mL混合血清样本的重复性精密度分别为2.60%、2.43%、0.91%,中间精密度分别为4.41%、3.36%、2.48%;HBV DNA国家标准物质GBW(E)090586(200 IU/mL)、GBW(E)090137(1.41×10~3 IU/mL)和GBW(E)090139(4.60×10~6 IU/mL)的实测值与靶值差异均在±0.4 Log_(10)IU/mL范围内;该系统在2.32×10~1～2.32×10~9IU/mL范围内呈线性(线性回归方程为Y=0.993 2X+0.161 2,R~2=0.998 9);该系统的定量限为30 IU/mL,检出限为10 IU/mL;干扰物质总胆红素(Tb)浓度≤512μmol/L、血红蛋白(Hb)浓度≤10 g/L、甘油三酯(TG)浓度≤18 mmol/L时,均对检测结果没有明显影响(偏倚±7.5%);交叉防污染实验中阳性标本检出率为100%,阴性标本检出率为0,全部阴性样本未受污染;不同批次试剂间检测结果差异符合要求(偏倚±7.5%);磁珠法提取的核酸室温放置2 h,再进行加样扩增,对检测结果没有明显影响(偏倚±7.5%)。结论:天隆HBV DNA超敏定量检测试剂盒在全自动荧光定量PCR仪cobas~?Z480上的检测性能基本符合临床应用要求。     

两种国产乙型肝炎病毒核酸定量检测试剂的检测效能评价

比较两种国产不同核酸提取方法定量检测乙型肝炎病毒（HBV）核酸试剂的检测效能。方法选择经抗病毒治疗且HBV DNA 载量在﹤1×10^4 IU/ml的乙型肝炎患者血清标本36份,采用两种国产HBV核酸定量检测试剂盒平行检测HBV DNA,对阳性血清进行梯度稀释后再检测,从定量线性范围、准确性、灵敏度、特异性等方面比较两种试剂的差异。结果在36例临床血清中,14份经科华试剂检测的结果为﹤500 IU/ml,而圣湘试剂检测的结果仍﹥1.00×10^3 IU/ml;对其中获得检测数据的31份标本进行两种试剂检测结果的相关性分析,发现一致性较好（r=0.817,P﹤0.05）;两种方法检测乙型肝炎患者血清HBV DNA的阳性率分别为55.6％和94.4％,差异具有统计学意义（x2=12.07,P=0.000）;对强阳性血清进行梯度稀释后定量检测显示,两种试剂检测水平的平均值与理论水平的线性相关性较好（湖南圣湘r=0.999,上海科华r=0.992）,但圣湘所有检测的相对偏差均在±0.3logIU/ml之内,而科华有两次检测的相对偏差超出了±0.3logIU/ml范围,提示圣湘试剂检测结果更稳定,使用纳米磁珠核酸提取法的检测结果较煮沸法更加准确。结论以纳米磁珠为提取核酸方法不仅具有更广的线性范围,同时可显著提高国产HBV DNA检测试剂的灵敏度和准确性。     

时间分辨荧光免疫分析法和酶联免疫吸附实验法测定乙型肝炎病毒大蛋白的方法学比较

目的对时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)和酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测乙型肝炎患者血清中病毒大蛋白(hepatitis B virus large surface protein,HBV-LP)进行方法学比较。方法收集30例正常体检血清标本作为阴性对照和150例慢性乙型肝炎患者血清标本,利用ELISA法检测HBV-LP,将其中70例阳性标本作为阳性样本,70例阴性标本作为阴性样本,再分别采用TRFIA法检测HBV-LP,对二者的灵敏度、特异度、一致性、检测线性范围、精密度、相关性及两种试剂盒的稳定性进行比较。其中TRFIA与ELISA结果不一致的12例标本采用PCR法进行验证。结果TRFIA检测HBV-LP的最小测定值为0.1 ng/ml,ELISA检测HBV-LP的最小测定值为2.5 ng/ml,二者的特异度均为100%。TRFIA和ELISA检测HBV-LP的Kappa值为0.83。12例ELISA和TRFIA检测不一致的标本,经PCR验证后有10例HBV DNA103拷贝数。TRFIA试剂检测的线性范围在0.625~10 252 ng/ml之间,ELISA试剂盒检测的线性范围在5~1 281.6 ng/ml。TRFIA法检测高、中、低3个浓度水平的批内CV平均为6.10%,ELISA法的批内CV平均为8.98%。TRFIA批间CV平均为6.91%,ELISA的批间CV平均为10.45%。TRFIA试剂盒的结合下降率为6.61%,ELISA试剂盒的结合下降率为23.59%。结论 TRFIA与ELISA具有高度的一致性,但是两者相比,前者有更高的灵敏度和精密度,且TRFIA试剂盒的稳定性更好。     

基于Probit回归的核酸检测系统分析灵敏度验证

目的:基于Probit回归,计算核酸血液检测系统所能检测到的分析物最低浓度,以验证核酸检测试剂在血站实验室常规使用中的分析灵敏度与试剂说明书中声明的一致性,保证核酸检测的质量。方法:使用核酸试剂对核酸系列分析灵敏度血清盘进行检测,统计HBV-DNA,HIV-RNA,HCV-RNA等每个浓度样本的重复检出率,应用SPSS统计学软件的Probit模块,计算出每个项目的95%检出限。结果:HBV-DNA的95%检出限为4.568IU/ml,HIV-RNA的95%检出限为46.000IU/ml,HCV-DNA 95%检出限为46.600IU/ml,均优于试剂说明书中声明的95%检出限。结论:基于Probit回归,使用核酸试剂对核酸系列分析灵敏度血清盘进行检测,能够准确计算出检测项目的95%检出限,直观判断实验室分析灵敏度是否在可接受范围之内。     

基于Probit回归的核酸检测系统分析灵敏度验证

目的:基于Probit回归,计算核酸血液检测系统所能检测到的分析物最低浓度,以验证核酸检测试剂在血站实验室常规使用中的分析灵敏度与试剂说明书中声明的一致性,保证核酸检测的质量。方法:使用核酸试剂对核酸系列分析灵敏度血清盘进行检测,统计HBV-DNA,HIV-RNA,HCV-RNA等每个浓度样本的重复检出率,应用SPSS统计学软件的Probit模块,计算出每个项目的95%检出限。结果:HBV-DNA的95%检出限为4.568IU/ml,HIV-RNA的95%检出限为46.000IU/ml,HCV-DNA 95%检出限为46.600IU/ml,均优于试剂说明书中声明的95%检出限。结论:基于Probit回归,使用核酸试剂对核酸系列分析灵敏度血清盘进行检测,能够准确计算出检测项目的95%检出限,直观判断实验室分析灵敏度是否在可接受范围之内。     

四种国产乙型肝炎表面抗原定量检测试剂对弱阳性样本检测结果的一致性比较

目的 比较4种国产HBsAg定量检测试剂与ARCHITECT i2000免疫检测系统对HBsAg弱阳性样本检测结果的一致性及检测值之间的相关性.方法 采用4种国产发光法检测系统和i2000检测系统分别检测185例HBsAg弱阳性血清标本以及不同浓度HBsAg的标准品.应用描述性统计和相关分析计算不同试剂分析结果的精密度、一致性和相关性.结果 4种国产发光法检测系统对i2000检测系统定量为0.05～1.00 IU/ml的弱阳性结果判断的符合率分别为25.93%、35.19%、51.85%和18.52%;对定量为＞1.00～10.00IU/ml的弱阳性结果判断的符合率分别为71.76%、87.79%、95.42%和69.47%.4种国产试剂均检测阴性的标本为i2000检测值在0.05～0.80IU/ml的低值阳性标本;i2000检测结果≥7.93 IU/ml的样本,4种国产发光法检测系统符合率为100%.国产试剂检测结果之间的相关性分析结果显示,其相关系数为0.8629～0.9265.结论 国产检测系统的分析灵敏度略低于i2000检测系统,检测值≤0.80IU/ml的样本不能给出与i2000一致的检测结果;对于≥7.93 IU/ml的样本,国产试剂均可给出与i2000一致的结果.

Abstract：

Objective To evalue the coincidence and correlation between the four domestic quantity assay reagents and with ARCHITECTi2000 immunoassay system.Methods 185 weak-reactive serum samples and standard materials of different concentrations were tested by four domestic quantity assay reagents for HBsAg test and ARCHITECTi2000 immunoassay system.The coincidence,the precision and the correlations between different systems were analyzed.Results The coincidence rates of the results of 0.05-1.00 IU/ml samples between the four domestic quantity assay reagents and ARCHITECTi2000 immunoassay system were 25.93%,35.19%,51.85% and 18.52% respectively,and for those results of ＞ 1.00-10.00 IU/ml samples the coincidence rates were 71.76%,87.79%,95.42% and 69.47% respectively.The samples of 0.05-0.80 IU/ml weak-reactive serum samples detected by the i2000 system were all negative detected by the four domestic systems.The coincidence rates of＞7.93 IU/ml serum samples detected by i2000 system were 100%detected by the four domestic systems.The correlations of the four domestic quantity assays were around 0.8629-0.9265.Conclusions The analysis sensitivity of the four domestic quantity assay reagents were below the i2000 system.The results of under 0.80 IU/ml samples detected by i2000 system were disaccord with the results detected by the four domestic systems,whereas for the sapmples over 7.93 IU/ml the results were consistent.     

两种乙型肝炎病毒核酸定量检测试剂的检验重复性与一致性比较

目的评估罗氏与国产乙型肝炎病毒核酸定量检测试剂的检验重复性与一致性分析,为临床应用提供参考。方法采用原装进口罗氏与国产乙型肝炎病毒核酸定量检测试剂盒检测滴度为60、170、1 700、17 000 IU/mL的WHO标准HBV DNA样本各20次,评估两种试剂的可溯源性和可重复性。再检测曾经或正在接受乙肝抗病毒治疗的患者9组(HBV DNA载量分别为50、50~200、~102、~103、~104、~105、~106、~107、~108IU/mL),每组20例,评估两种试剂的一致性。结果 WHO标准HBV DNA样本检测得到罗氏检测试剂ICC值为0.93(F=12.51,P0.0001),国产检测试剂为0.82(F=8.72,P0.0001),可得罗氏检测重复性优于国产组。通过两组临床血清检测的检测结果进行相关性分析结果得两者相关系数r=0.849,P0.0001,相关性良好。两组临床血清检测的Bland-Altman图,可见两种检测试剂结果差值90%的位点位于95%置信区间参考范围内,说明两种检验方法间一致性良好。结论罗氏乙型肝炎病毒核酸定量检测试剂的检测结果与国产试剂相比,一致性良好,但罗氏乙型肝炎病毒核酸定量检测试剂的重复性优于国产试剂,是乙肝患者抗病毒治疗过程中病毒载量监控的金标准。     

HIV-1 DNA评价盘的构建及两种国产检测试剂性能评价

目的构建1型艾滋病病毒(HIV-1)脱氧核糖核酸(DNA)全血和干血斑评价盘,并对两种国产HIV-1 DNA检测试剂(试剂A和试剂B)性能进行实验室评价。方法从中国疾病预防控制中心性病艾滋病预防控制中心参比实验室样本库中筛选全血和干血斑样本,构建HIV-1 DNA全血和干血斑评价盘,评价盘包含阳性参考品20份,阴性参考品20份,最低检测限参考品5份,精密度参考品2份;并从云南省德宏州收集临床样本,通过评价盘和临床样本对两种试剂的敏感性、特异性、最低检测限和精密度进行评价。结果从云南省德宏州疾病预防控制中心和佑安医院共收集临床样本278份(全血和对应干血斑各278份),包含207份阳性样本和71份阴性样本。阳性样本中,106份未接受抗病毒治疗(ART),101份已接受ART。全血评价盘结果显示,两种试剂的阴、阳性样本符合率20/20,最低检测限均为4/5,A试剂精密度为1.2%(CV1)和1.4%(CV2),B试剂精密度为2.8%(CV1)和1.3%(CV2)。干血斑评价盘结果显示,两种试剂阴、阳性样本符合率均为20/20,最低检测限均为3/5,A试剂精密度为2.5%(CV1)和3.4%(CV2),B试剂精密度为3.1%(CV1)和2.8%(CV2)。177份未进行ART临床样本评价结果显示,对于全血样本,两种试剂特异性和敏感性均为100%(71/71,106/106);对于干血斑样本,两种试剂特异性均为100%(71/71),试剂A敏感性为98.1%(104/106),试剂B敏感性为99.1%(105/106)。101份已接受ART样本评价结果显示,对于全血样本,试剂A和试剂B敏感性均为100%(101/101);对于干血斑样本,试剂A敏感性为91.1%(92/101),试剂B敏感性为90.1%(91/101)。结论两种HIV DNA检测试剂均能符合全血和干血斑评价盘要求;在检测未进行ART全血和干血斑临床样本时均表现良好,且试剂A与B之间无显著差异,两种国产HIV-1 DNA检测试剂均能满足HIV-1核酸检测要求。     

不同商品化试剂对临床血清样本HBV DNA定量检测结果的影响

目的:评价不同商品化试剂定量检测临床血清样本乙型肝炎病毒DNA(HBV DNA)性能的差异。方法:(1)应用5种商品化HBV DNA定量试剂(2种进口试剂A和B,3种国产试剂C、D和E)分别对6例混合血清样本进行检测,并对结果进行比较;3种国产试剂对6例混合血清样本以及10~3和10~6两个水平的HBV DNA国家标准物质GBW(E)090137和GBW(E)090139分3个批次检测,分析其精密度和正确度。(2)采用3种国产试剂定量检测43例慢性乙型肝炎患者血清样本HBV DNA,并分析其相关性和一致性。结果:(1)定量检测6例混合血清样本中HBV DNA,试剂B、C与A的检测结果差异均无统计学意义(P0.05),而试剂D、E的检测结果均显著低于试剂A(P0.05)。(2)定量检测6例混合血清样本以及10~3和10~6两个水平的HBV DNA国家标准物质,3种国产试剂的CV均小于5%,国家标准物质的实际检测值与靶值的差值绝对值均小于0.4 log_(10) IU/mL,精密度和正确度满足行业标准。(3)对于43例临床血清样本,试剂C、D和E的阳性检出率分别为95.35%(41/43)、95.35%(41/43)、86.05%(37/43);对于检测结果均在3种试剂定量范围内的24例临床血清样本,HBV DNA定量检测结果为CDE(P0.05),3种试剂的检测结果两两间均呈线性相关(C vs D:R~2=0.93,Y=0.973X-0.164;C vs E:R~2=0.61,Y=0.770X+0.210;D vs E:R~2=0.69,Y=0.809X+0.270),试剂C和D、C和E、D和E的Bland-Altman分析发现其检测结果差值平均值分别为0.28、0.80和0.51 log_(10) IU/mL,相应的95%一致性界限分别为(-0.30,0.86)、(-0.62,2.21)、(-0.74,1.77),相应的95%一致性界限以内的点分别占95.83%(23/24)、95.83%(23/24)和91.67%(22/24),其一致性界限范围内检测值的最大差异分别为0.85、1.96和1.25 log_(10)IU/mL。试剂C和D、C和E、D和E的检测结果差值1.0 log_(10) IU/mL的血清样本分别占0(0/24)、33.33%(8/24)和25.00%(6/24)。结论:满足行业标准的不同商品化国产试剂定量检测临床血清样本HBV DNA的性能存在显著差异,在常规临床实践中互换用于临床决策时务必慎重。     

3种HBsAg血液筛查试剂的检测效能评估

目的:对使用的3种HBsAg(2种酶免试剂+1种化学发光试剂)进行检测效能评估。方法:通过分析这3种HBsAg试剂对9个HBsAg血清盘(792个标本)的检测结果,分析3种试剂的批间精密度、批内精密度及其灵敏度、特异度及其正确率、假阳性例数和假阴性例数,最终比较3种试剂检测效能。结果:对于强阳性质控标本,3种试剂的批间精密度良好,变异系数在5.1%～10.2%。由于ELISA2对于弱阳性质控标本未能检出,因此ELISA2在批间精密度和批内精密度的统计中不计入内。对于弱阳性质控,ELISA1与CLIA的批间精密度分别为16.2%和21.7%。批内精密度的变异系数分别为10.9%和12.6%。对于亚型标本的分析,ayw1亚型的标本ELISA试剂1的检出下限是0.18IU/mL,ELISA试剂2的检出下限是0.36IU/mL,CLIA试剂的检出下限是0.09IU/mL。adw2亚型的标本ELISA试剂1的检出下限是0.09IU/mL,ELISA试剂2的检出下限是0.09IU/mL,CLIA试剂的检出下限是0.05IU/mL。adrq亚型的标本ELISA试剂1的检出下限是0.05IU/mL,ELISA试剂2的检出下限是0.09IU/mL,CLIA试剂的检出下限是0.05IU/mL。对于突变标本,3种试剂的检出率为42.3%～88.5%,其中CLIA检出率为88.5%。在611例常规血清盘标本,3种HBsAg试剂灵敏度为78.7%～87.5%,特异性为96.3%～98.4%,准确率为84.9%～90.2%,其中ELISA2的正确率最低为84.9%,假阳性例数为3～6例,假阴性例数为41～88例,其中CLIA的假阴性例数最少为6例。结论:CLIA试剂对于突变标本和亚型标本具有很好的检出能力。HBsAg试剂在使用前及使用中均要进行必要的评估,化学发光试剂在HBsAg检测中具有一定优势。     

贝氏柯克斯体实时荧光定量PCR方法的建立及对云南鼠标本检测

目的 建立贝氏柯克斯体荧光定量PCR方法并应用于云南省鼠标本的检测.方法 根据贝氏柯克斯体ISlllla基因设计引物和探针,以克隆的ISlllla基因片断(485 bp)作标准DNA模板,建立实时荧光定量PCR检测方法,并与普通巢式PCR方法进行比较.采用2种方法对云南玉溪自然界采集的鼠各类标本进行检测分析,计算2种方法的检出率.结果 实时荧光定量PCR标准曲线的循环阈值(cycle threshold,Ct)与模板拷贝数呈良好的线性关系(r=-0.999),重复性测试Ct变异系数(coefficient of variation,CV)为0.25%～3.98%,灵敏度为巢式PCR的10倍.以其他立克次体和常见病原菌共26种DNA为模板进行特异性检测,结果均为阴性.实时荧光定量PCR检测鼠血、肝脏、脾脏中的贝氏柯克斯体,阳性率分别为32.31%、61.29%、85.19%;巢式PCR法检测相应标本阳性率分别为27.69%、11.29%、74.07%;间接免疫荧光试验检测鼠血清中贝氏柯克斯体IgG抗体阳性率为26.15%.结论 本实验建立的实时荧光定量PCR比巢式PCR敏感,且具有良好的特异性,可用于人群和动物贝氏柯克斯体感染监测及临床标本的检测.     

An objective computer system for the quantification of artery stenoses

Summary We have developed a low cost, clinically usable system for the objective assessment of the severity of coronary artery stenoses from single view angiograms. The system is based on a desktop computer with incorporated frame grabber. Images are captured by means of a video camera.The user selects a region of interest which encompasses the stenosis. Facilities are provided for automatic or manual definition of the artery centre line and edges. The computer then calculates the artery diameter and cross-sectional area by videodensitometry along profile lines which are orthogonal to the long axis of the artery. These results can be expressed numerically as a percentage stenosis when compared to a normal region of the artery.The image is corrected for geometric distortion using a grid test object. The image grey scale is corrected by means of a ramp test object such that a pixel value is proportional to the attenuator thickness. The ramp is placed on the patient during the X-ray examination and an iterative technique has been developed for subtracting the underlying structures from the superimposed ramp image. The system has been assessed using test objects constructed in Perspex which simulate arteries of known cross-sectional area and stenoses of known severity.     

Persistence of social signatures in human communication

The social network maintained by a focal individual, or ego, is intrinsically dynamic and typically exhibits some turnover in membership over time as personal circumstances change. However, the consequences of such changes on the distribution of an ego’s network ties are not well understood. Here we use a unique 18-mo dataset that combines mobile phone calls and survey data to track changes in the ego networks and communication patterns of students making the transition from school to university or work. Our analysis reveals that individuals display a distinctive and robust social signature, captured by how interactions are distributed across different alters. Notably, for a given ego, these social signatures tend to persist over time, despite considerable turnover in the identity of alters in the ego network. Thus, as new network members are added, some old network members either are replaced or receive fewer calls, preserving the overall distribution of calls across network members. This is likely to reflect the consequences of finite resources such as the time available for communication, the cognitive and emotional effort required to sustain close relationships, and the ability to make emotional investments.Social relationships play an important functional role in human society both at the collective level and by providing benefits to individuals. In particular, it appears that having strong and supportive relationships, characterized by closeness and emotional intensity, is essential for health and well-being in both humans and other primates (1, 2). At the same time, there is a higher cost to maintaining closer relationships, reflected in the amount of effort required to maintain a relation at the desired level of emotional closeness. Because of this, the number of emotionally intense relationships is typically small. Moreover, it has been suggested that ego networks, the sets of ties individuals (egos) have to their friends and family (alters), may be subject to more general constraints associated with limits on human abilities to interact with large numbers of alters (3–5). Although there are obvious constraints on the time available for interactions (5–7), additional constraints may also arise through limits on memory capacity (3, 8) or other cognitive abilities (9, 10).Irrespective of the specific mechanisms that act to constrain ego networks, it is reasonable to ask whether such mechanisms shape these networks in similar ways under different circumstances, giving rise to some characteristic features that persist over time despite network turnover. Here, we explore this question with a detailed analysis of the communication patterns within ego networks in an empirical setting that results in large membership turnover and changes in the closeness of relationships. In particular, we focus on the way that egos divide their communication efforts among alters and how persistent the observed patterns are over time. We call these patterns, which may be expected to vary across individuals, social signatures.Over the last decade, research on human communication has been given a significant boost by the widespread adoption of new communication technologies. The popularity of communication channels such as mobile phones and online environments has made it possible to capture microlevel quantitative data on human interactions automatically, in a way that circumvents biases inherent in retrospective self-reports (11). However, studies using electronic communication sources typically lack information on the nature of the social relationships (5, 12–15), whereas the challenge in using survey data alone has been that these give detailed information about the nature of the social relationships, but lack quantitative information about the actual patterns of communication (16). Further, in surveys the respondent burden from recording communication events with their entire ego network is very high (17) and people’s accuracy in recalling detailed communication events is known to be limited (18).We combine detailed, autorecorded data from mobile phone call records with survey data. These were collected during a study (19) that tracked changes in the ego networks of 24 students over 18 mo as they made the transition from school to university or work (details in Materials and Methods). These changes in personal circumstances result in a period of flux for the social relationships of the participants, with many alters both leaving and entering their networks. This provides a unique setting for studying network-level structure and its response to major changes in social circumstances. This dataset combines detailed data on communication patterns from mobile phone call records with questionnaire data that explore participants’ own perceptions of the quality of the relationships with all of the members of their network. More importantly, call record data contain complete time-stamped records of all calls made by egos to alters in their network, rather than just a subset of calls egos make to alters who happened to be on the same mobile network as they are (as has usually been the case in previous work, e.g., ref. 12). The questionnaires that augmented the call records provide information on the networks of participants that includes assessment of emotional closeness, time between face-to-face contact, and the phone numbers of alters. This allowed the call records of alters with several phone numbers (mobile phones, landlines) to be merged, giving a more accurate picture of communication between two individuals than that based on mobile phone calls alone.These data enable us to uncover changes in the structure of the ego networks of the participants, reflected in their communication behavior. We find a consistent pattern that is seen to be persistent over time even when there is large network turnover. This social signature is consistent with previously observed patterns of social network site use (20, 21) and text messaging (22, 23) in that a high proportion of communication is focused on a small number of alters. A detailed analysis of the social signatures of individual participants reveals that there is individual variation in the exact way their limited communication time is allocated across their network members. Although individual signatures show some response to network turnover, they surprisingly retain much of their distinctive variation over time despite this turnover.     

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ～1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.     

Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing

Real-time monitoring of stem cells (SCs) differentiation will be critical to scale-up SC technologies, while label-free techniques will be desirable to quality-control SCs without precluding their therapeutic potential. We cultured adipose-derived stem cells (ADSCs) on top of multielectrode arrays and measured variations in the complex impedance Z* throughout induction of ADSCs toward osteoblasts and adipocytes. Z* was measured up to 17 d, every 180 s, over a 62.5-64 kHz frequency range with an ECIS Z instrument. We found that osteogenesis and adipogenesis were characterized by distinct Z* time-courses. Significant differences were found (P = 0.007) as soon as 12 h post induction. An increase in the barrier resistance (Rb) up to 1.7 ohm·cm(2) was associated with early osteo-induction, whereas Rb peaked at 0.63 ohm·cm(2) for adipo-induced cells before falling to zero at t = 129 h. Dissimilarities in Z* throughout early induction (<24 h) were essentially attributed to variations in the cell-substrate parameter α. Four days after induction, cell membrane capacitance (Cm) of osteo-induced cells (Cm = 1.72 ± 0.10 μF/cm(2)) was significantly different from that of adipo-induced cells (Cm = 2.25 ± 0.27 μF/cm(2)), indicating that Cm could be used as an early marker of differentiation. Finally, we demonstrated long-term monitoring and measured a shift in the complex plane in the middle frequency range (1 kHz to 8 kHz) between early (t = 100 h) and late induction (t = 380 h). This study demonstrated that the osteoblast and adipocyte lineages have distinct dielectric properties and that such differences can be used to perform real-time label-free quantitative monitoring of adult stem cell differentiation with impedance sensing.     

定量血流分数评价左心室舒张功能不全患者心肌缺血的价值

目的探讨定量血流分数(quantitative flow ratio,QFR)在诊断左心室舒张功能不全患者心肌缺血的价值。方法纳入2017年1月至2018年12月间广东省人民医院心脏超声诊断为左心室舒张功能不全但收缩功能正常,同时行冠状动脉造影及血流储备分数(fractional flow reserve,FFR)检查的患者110例。根据FFR测量值分为心肌缺血组(FFR≤0.80,n=52)和对照组(FFR>0.80,n=58)。比较两组患者的临床资料,并分别用QFR与定量冠状动脉造影(quantitative coronary angiography,QCA)两种工具分析造影图像,绘制受试者工作曲线(receiver operating characteristic curve,ROC)并评价QFR的诊断效能。结果两组患者的临床资料比较,差异无统计学意义(P>0.05)。以FFR作为参考标准,QFR的诊断效能均明显高于QCA,其中准确率[83.6%(95%CI:75.5~89.5)vs. 59.1%(95%CI:49.7~67.8)]、敏感度[75.0%(95%CI:61.7~84.9)vs. 46.2%(95%CI:33.3~59.5)]、特异度[91.4%(95%CI:81.0~96.7)vs. 70.7%(95%CI:57.9~80.9)]、阳性预测值[88.6%(95%CI:75.6~95.5)vs.58.5%(95%CI:43.4~72.3)]及阴性预测值[80.3%(95%CI:69.0~88.3)vs. 59.4%(95%CI:47.6~70.2)]比较,差异均有统计学意义(P<0.01)。QFR与QCA的阳性似然比分别为8.721 vs. 1.577,阴性似然比分别为0.274 vs.0.761。QFR与QCA诊断心肌缺血的ROC的曲线下面积分别为0.94(95%CI:0.88~0.98)和0.67(95%CI:0.58~0.76)(Z=5.167,P<0.0001)。结论 QFR比QCA能更灵敏、早期识别导致左心室舒张功能不全患者的心肌缺血情况,在指导治疗、改善患者预后方面具有重要临床意义。