腹泻病毒化学发光基因芯片检测方法的建立和验证

目的 建立一种化学发光基因芯片检测方法,实现7种腹泻病毒,A组轮状病毒、B组轮状病毒、Ⅰ型诺如病毒、Ⅱ型诺如病毒、札如病毒、星状病毒和肠道腺病毒的快速、准确检测.方法 选择7种病毒特异性基因的保守区,设计引物与探针,制备寡核苷酸基因芯片.将多重实时荧光PCR(RT-PCR)扩增产物与带有特异性探针的芯片杂交,经洗涤、化学发光检测后进行结果分析.在优化的RT-PCR体系、杂交条件和化学发光检测条件下,评价芯片的灵敏度、重复性和特异性.结果 研制的基因芯片具有良好的特异性和灵敏度,检测体外转录RNA参考品的最低检测限为3×103拷贝/反应,检测临床样本的灵敏度为95.2％、特异性为92.1％、符合率为95.1％.结论 建立了一种基于化学发光基因芯片的腹泻病毒检测方法,此法能快速、灵敏、特异地检测和鉴别7种腹泻病毒,具有较好的应用前景.     

耐万古霉素肠球菌检测基因芯片方法的建立和验证

目的：基于基因芯片技术建立一种能快速甄别粪肠球菌、屎肠球菌及万古霉素耐药基因的检测方法。方法根据GenBank中查找的2种常见肠球菌特异性基因序列（ ddl）及万古霉素耐药基因（ vanA,vanB）序列,设计与合成用于检测肠球菌的特异性基因及耐药基因的引物和探针,制备耐万古霉素肠球菌（VRE）检测基因芯片。利用多重不对称PCR法扩增样品中的特异性基因与耐药基因片段,标记产物与基因芯片上的探针杂交,经清洗、化学发光法显色后进行结果分析。在优化的多重PCR体系、杂交反应和化学发光法检测条件下,评价芯片的特异性、灵敏性和重复性。结果共筛选出1对通用引物、4对特异性引物和1条细菌通用探针、4条特异性检测探针。该芯片具有良好的特异性和重复性,灵敏性可达103 CFU／ml。10例临床分离株样本的芯片检测结果与药敏实验基本一致（8／10）。结论初步建立了检测VRE的基因芯片方法,利用此方法可以甄别2种VRE种类并检测其耐药基因。     

可视化基因芯片技术检测肠道病毒方法的建立

目的建立一种能同时检测10种肠道病毒的可视化基因芯片法。方法根据公开发表的10种常见肠道病毒的序列,设计病毒引物和探针,制备肠道病毒检测基因芯片。利用多重不对称PCR法扩增样品中的病毒靶片段,标记产物与基因芯片上的探针杂交,经清洗、可视化显色后进行结果分析。在优化的RT-PCR体系、杂交反应和可视化检测条件下,评价芯片的特异性、灵敏度和重复性。结果本研究共筛选出2对通用引物、3对特异性引物和1条肠道病毒属通用探针、9条特异性检测探针。该芯片具有较好的特异性和重复性,可检测出不低于102拷贝/μl的体外转录RNA。30例临床标本的芯片检测结果与荧光PCR法一致。结论本研究所建立的方法具有高通量、高特异性、高灵敏度等特点,因此在临床上具有潜在的应用前景,可以为肠道病毒诊断提供实验室依据。     

可视化基因芯片技术检测肠道病毒方法的建立

目的建立一种能同时检测10种肠道病毒的可视化基因芯片法。方法根据公开发表的10种常见肠道病毒的序列,设计病毒引物和探针,制备肠道病毒检测基因芯片。利用多重不对称PCR法扩增样品中的病毒靶片段,标记产物与基因芯片上的探针杂交,经清洗、可视化显色后进行结果分析。在优化的RT-PCR体系、杂交反应和可视化检测条件下,评价芯片的特异性、灵敏度和重复性。结果本研究共筛选出2对通用引物、3对特异性引物和1条肠道病毒属通用探针、9条特异性检测探针。该芯片具有较好的特异性和重复性,可检测出不低于102拷贝/μl的体外转录RNA。30例临床标本的芯片检测结果与荧光PCR法一致。结论本研究所建立的方法具有高通量、高特异性、高灵敏度等特点,因此在临床上具有潜在的应用前景,可以为肠道病毒诊断提供实验室依据。     

多种食源性致病菌的基因芯片检测技术

目的探讨随机PCR结合基因芯片技术用于多种食源性致病菌筛查检测的可行性。方法利用Clustal X和Oligo 6.0软件设计探针,进行生物信息学比对验证特异性,探针末端修饰后合成并制备基因芯片。使用锚定随机引物扩增细菌基因组DNA,偶联荧光染料的扩增产物与芯片杂交后扫描检测。对随机PCR及杂交反应等一系列检测条件进行优化。选取16种病原细菌进行芯片特异性验证,使用4种食源性致病菌DNA进行芯片灵敏度检测及重复性评价,制备细菌DNA混合样本和痢疾志贺菌模拟水污染样本对芯片检测效能进行初步考核。结果 9种食源性致病菌获得阳性结果,基因组DNA检测灵敏度102～103pg/μl,芯片重复性变异系数(CV)值<15%,混合样本检测与预期结果一致,最低可检测水样本中痢疾志贺菌的最低浓度为3.54×105cfu/ml。结论初步建立的基于随机引物PCR的基因芯片技术进行多种食源性致病菌检测的方法,为病原细菌高通量筛查检测提供了一种新的思路。     

基于随机聚合酶链反应的病原菌基因芯片检测技术

目的建立一种基于随机聚合酶链反应的病原细菌基因芯片筛查检测技术。方法用生物学软件分析7种病原菌特异性基因序列的保守性区域,利用Oligo 6.0软件设计针对靶细菌的一系列探针,制备检测用基因芯片。细菌基因组DNA在随机引物扩增中掺入氨基丙烯-dUTP,产物偶联荧光染料后与芯片上探针杂交,通过芯片扫描仪和图像分析软件对结果进行判断。选取19种病原菌基因组DNA进行芯片特异性验证和灵敏度评价,使用问号钩端螺旋体对应靶探针进行基因芯片检测方法重复性验证,并制备问号钩端螺旋体模拟水污染样本进行芯片检测。结果在均一的杂交条件下4种靶细菌均能得到相应特异性杂交图谱,其他非目的细菌均为阴性结果,3种靶细菌基因组DNA最低检测浓度为14.43～363.4 pg/μl,芯片重复性变异系数CV值<15%,最低可检测含问号钩端螺旋体7×105条/ml模拟水污染样本。结论初步建立的随机聚合酶链反应结合芯片技术的检测方法可用于多种病原菌筛查检测,为细菌高通量筛查与鉴定技术提供了新的思路和实验依据。     

实时荧光定量PCR检测莫氏立克次体

目的 采用TaqMan-MGB探针建立检测莫氏立克次体的实时荧光定量PCR.方法 依据莫氏立克次体外膜蛋白B基因(ompB)设计引物和探针,以克隆的ompB作模板建立实时荧光定量PCR方法.结果 建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.995);该方法能检出＜10拷贝的莫氏立克次体DNA,敏感性为普通PCR的1 000倍.用该方法检测莫氏立克次体DNA,结果为阳性,但是检测其他相关细菌DNA的结果均为阴性.用该方法检测莫氏立克次体感染豚鼠血标本,50%样本检测为阳性,而普通PCR检测结果均为阴性.结论 该实时荧光定量PCR具有很高的特异性和敏感性,适合于快速检测样本中微量莫氏立克次体DNA,可用于临床实验室快速确诊地方性斑疹伤寒.     

基于随机聚合酶链反应的病原菌基因芯片检测技术

目的建立一种基于随机聚合酶链反应的病原细菌基因芯片筛查检测技术。方法用生物学软件分析7种病原菌特异性基因序列的保守性区域,利用Oligo 6.0软件设计针对靶细菌的一系列探针,制备检测用基因芯片。细菌基因组DNA在随机引物扩增中掺入氨基丙烯-dUTP,产物偶联荧光染料后与芯片上探针杂交,通过芯片扫描仪和图像分析软件对结果进行判断。选取19种病原菌基因组DNA进行芯片特异性验证和灵敏度评价,使用问号钩端螺旋体对应靶探针进行基因芯片检测方法重复性验证,并制备问号钩端螺旋体模拟水污染样本进行芯片检测。结果在均一的杂交条件下4种靶细菌均能得到相应特异性杂交图谱,其他非目的细菌均为阴性结果,3种靶细菌基因组DNA最低检测浓度为14.43～363.4 pg/μl,芯片重复性变异系数CV值〈15%,最低可检测含问号钩端螺旋体7×105条/ml模拟水污染样本。结论初步建立的随机聚合酶链反应结合芯片技术的检测方法可用于多种病原菌筛查检测,为细菌高通量筛查与鉴定技术提供了新的思路和实验依据。     

基因芯片技术鉴定出血性大肠杆菌O157∶H7和霍乱弧菌O139

目的:建立一种同时快速检测肠出血性大肠杆菌(EHEC)O157∶H7和霍乱弧菌O139的基因芯片,并验证该芯片的特异性和敏感性。方法:选择EHEC O157∶H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因(u idA);霍乱弧菌O139的肠毒素A亚单位(ctxA)、毒力协调菌毛A亚单位(tcpA)、糖基转移酶(glycosotransferaseLPSgt)基因序列分别设计引物和探针,反向引物用荧光素Cy3标记,探针在3′端氨基修饰。在优化的PCR和杂交反应条件下,分别进行三重PCR扩增,产物混合后与芯片进行杂交,产生特异性荧光信号,进一步筛选探针。随后将筛选出的探针制备芯片用于检测临床样本。结果:PCR产物在相应探针处均产生特异性杂交信号,临床样本检测结果表明,此芯片比常规细菌学检测方法灵敏。结论:所研制的同时定性检测EHEC O157∶H7和霍乱弧菌O139的基因芯片是特异、灵敏而且快速的,为这两种肠道致病菌感染的快速诊断提供了新的方法。     

基因芯片技术鉴定出血性大肠杆菌O157:H7和霍乱弧菌O139

目的：建立一种同时快速检测肠出血性大肠杆菌（EHEC）O157：H7和霍乱弧菌O139的基因芯片,并验证该芯片的特异性和敏感性.方法：选择EHEC O157：H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因（uidA）;霍乱弧菌O139的肠毒素A亚单位（ctxA）、毒力协调菌毛A亚单位（tcpA）、糖基转移酶（glycosotransferase LPSgt）基因序列分别设计引物和探针,反向引物用荧光素Cy3标记,探针在3′端氨基修饰.在优化的PCR和杂交反应条件下,分别进行三重PCR扩增,产物混合后与芯片进行杂交,产生特异性荧光信号,进一步筛选探针.随后将筛选出的探针制备芯片用于检测临床样本.结果：PCR产物在相应探针处均产生特异性杂交信号,临床样本检测结果表明,此芯片比常规细菌学检测方法灵敏.结论：所研制的同时定性检测EHEC O157：H7和霍乱弧菌O139的基因芯片是特异、灵敏而且快速的,为这两种肠道致病菌感染的快速诊断提供了新的方法.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.