原料血浆和凝血因子类产品中人细小病毒B19污染情况调查

目的：检测混合原料血浆和凝血因子类产品中人细小病毒B19（human parvovirus B19,B19病毒）核酸,分析我国原料血浆及凝血因子类产品中B19病毒的污染情况。方法针对B19病毒的保守区域NS1区合成引物及探针,采用TaqMan实时定量PCR法检测混合原料血浆和4类凝血因子类产品中B19病毒核酸。结果混合原料血浆的B19病毒核酸的阳性率为59．49％（116／195）,最高浓度可达1．35×1010拷贝／ml。因子Ⅷ、凝血酶、纤维蛋白原以及凝血酶原复合物的阳性率分别为93．55％（29／31）、100％（10／10）、85．71％（6／7）和88．89％（8／9）。结论我国混合原料血浆与凝血因子类产品中B19病毒的污染率较高,有必要进行原料血浆的B19病毒筛查,对于保障血液制品的病毒安全性具有重要意义。     

7种立克次体甄别检测基因芯片方法的建立

目的：建立一种能同时检测7种立克次体的化学发光基因芯片法。方法根据NCBI公开发表的7种立克次体的序列设计引物和探针,制备立克次体甄别检测基因芯片。利用多重不对称PCR法扩增立克次体靶基因片段,标记的产物与基因芯片上的探针杂交,经清洗、化学发光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检测条件下,评价芯片的特异性、灵敏度、重复性。用实时荧光PCR法与芯片法分别检测莫氏立克次体梯度稀释的核酸,比较两种方法的灵敏度。制备双盲模拟样本,进一步评价芯片方法的准确性。结果该研究共筛选出1对通用引物、4对特异性引物和1条立克次体属通用探针、9条特异性检测探针。该芯片检测质粒DNA的灵敏度为1．5×102～3×103拷贝／反应,检测模拟样本的灵敏度为103～104拷贝／μl。实时荧光PCR法与芯片法检测结果一致,实时荧光PCR法比芯片法灵敏度高10倍。双盲模拟样本检测符合率为100％。结论成功建立了可同时检测7种立克次体的化学发光基因芯片检测方法,为立克次体病的临床诊断和流行病学调查提供了一种新的高通量检测手段。     

基于重组酶聚合酶扩增结合侧流层析技术的B、E型腺病毒快速可视化检测方法的研究与评价

目的 建立一种快速、准确、可视化、易开展的B、E型腺病毒检测技术.方法 针对B、E型腺病毒壳蛋白保守序列设计通用引物,建立可同时检测出这两种腺病毒型别的重组酶聚合酶扩增结合侧流层析试纸条(recombinase polymerase amplification combined with a lateral flow dipstick,LFD-RPA)检测技术,评价其灵敏度与特异性,并验证最佳反应温度与反应时间,同时利用该法对19例B、E型腺病毒感染患者及10例健康志愿者鼻咽拭子标本进行检测.结果 腺病毒LFD-RPA法检测灵敏度可达10拷贝/μl,与qPCR方法相近,且不会与其他亚属腺病毒及其他病毒发生交叉反应.反应可在25~45℃温度范围内高效进行,检测全程仅需15~20 min.利用临床咽拭子标本对检测体系进行评估,其灵敏度及特异性均达100%.结论 该检测方法灵敏度高、特异性强、操作简单,反应快速,能脱离对大型仪器、专业操作人员及正规实验室的依赖,在基层卫生部门,尤其在野外及现场检测中具有极大的应用潜力.     

森林脑炎病毒(TBEV)实时定量TaqMan PCR检测方法的建立

目的建立快速检测TBEV的实时定量TaqMan PCR方法。方法根据GenBank发表的TBEV全基因组序列资料,在其C基因和NS5基因区段设计TBEV的特异探针和引物,在E基因区段设计普通PCR引物。以MDJ01株作为待检毒株,黄病毒属的另外7株病毒用来评价检测体系的特异性。测定病毒的TCID50值并制备病毒拷贝数标准品,分别用于制作病毒滴度和拷贝数标准曲线。与常规PCR方法进行了灵敏度比较,并建立了病毒感染小鼠的检测模型。结果该检测方法的灵敏度可达到100拷贝／反应或0．1TCID50,是常规PCR方法的十倍。作为对照的黄热病毒疫苗株17D、登革1～4型病毒、日本脑炎病毒、西尼罗病毒Chin-01株结果均为阴性,证明该体系具有良好的特异性。经过多批次、不同浓度样品的重复检测,批内和批间变异系数均小于5％,表明该体系具有较好的稳定性和重复性。结论建立了一种灵敏、特异、简便易行的TBEV的TaqMan实时定量PCR检测方法,为TBEV的预防控制和诊断提供了一种技术手段。     

荧光定量PCR快速检测金黄色葡萄球菌方法的建立

目的利用SmartCycler系统建立一种简便、快速的荧光定量PCR检测金黄色葡萄球菌（Staphylococcus aureus,简称SAU）的方法。方法根据SAU特异nuc基因设计引物和探针,建立检测体系,并在体系中加入内质控IAC,通过另一个标记不同荧光基团的TaqMan探针来监测IAC。用SAU6538株污染饮用水和牛奶模拟实际样本,以评价体系的性能。结果以含nuc片段的线性质粒为模板,反应体系的灵敏度为5拷贝/μl;以6538株基因组DNA为模板,最低检测限为10fg/μl;用涂平板法计数并10倍梯度稀释的菌液提取DNA为模板,最低检测限为50cfu/ml。建立的定量标准曲线的循环域值（Ct）与模板拷贝数呈良好的线性关系（r2≥0.998）,PCR效率E≥96.7%。用SAU6538污染饮用水和牛奶模拟实际样本,灵敏度可达5×102cfu/25ml样本。结论所建立的nuc-IAC荧光定量PCR具有内质控IAC,既能定量检测食物是否被污染,又能监测PCR体系,防止假阴性发生或低估病原菌污染量。     

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。     

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.     

实时RT-PCR检测甲型H3N2流感病毒R292K耐药位点

目的建立快速检测甲型H3N2流感病毒神经氨酸酶（NA）奥司他韦耐药突变位点的实时RT-PCR方法。方法通过设计特异性靶向NAR292K突变位点的Taq-MGB探针进行实时RT-PCR反应,并利用模拟样本和临床标本进行灵敏性和特异性评价。结果与结论建立了快速检测NAR292K位点的实时RT-PCR检测方法;本方法灵敏性高,可检测低至500拷贝/μl;特异性好,与无耐药突变的甲型H3N2流感病毒及其他呼吸道病毒均无交叉反应,为流感耐药监测提供了有效工具。     

基于多重PCR的呼吸道病毒纳米孔测序快速检测的方法学研究

目的 通过对多重PCR扩增条件的优化和纳米孔测序中病原微生物检测特异性及灵敏度的评价,建立一种由多重PCR和纳米孔测序技术相结合的检测技术体系,满足对呼吸道病毒现场快速检测鉴定的需求。方法 首先针对呼吸道病毒设计特异性引物,其次优化PCR扩增条件,建立快速测序体系,最后对不同浓度的病原体进行扩增与测序鉴定,评价检测体系的灵敏度。结果 优化后的多重PCR检测体系在退火温度为55℃、引物添加浓度为0.1μmol/L时,可同时检出25种呼吸道病毒,检测灵敏度可达1×10⁴拷贝/ml,整体检测时间在4 h以内。结论 该方法简单快捷,能够实时获取扩增片段的序列信息,特异性强,灵敏度较高,有望为呼吸道病原体的快速筛选和现场检测鉴定提供有力技术支撑。     

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.     

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).     

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.     

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.     

Prevalence and severity of hip and groin pain in sub‐elite male football: a cross‐sectional cohort study of 695 players

The purpose of this study was twofold: (a) to investigate the prevalence of hip and groin pain in sub‐elite male adult football in Denmark and (b) to explore the association between prevalence and duration of hip and groin pain in the previous season with the Copenhagen Hip and Groin Outcome Score (HAGOS) in the beginning of the new season. In total 695 respondents from 40 teams (Division 1–4) were included. Players completed in the beginning of the new season (July–Sept 2011) a self‐reported paper questionnaire on hip and/or groin pain during the previous season and HAGOS. In total 49% (95% CI: 45–52%) reported hip and/or groin pain during the previous season. Of these, 31% (95% CI: 26–36%) reported pain for >6 weeks. Players with the longest duration of pain during the previous season had the lowest HAGOS scores, when assessed at the beginning of the new season, P < 0.001. This study documents that half of sub‐elite male adult football players report pain in the hip and/or groin during a football season. The football players with the longest duration of pain in previous season displayed the lowest HAGOS scores in the beginning of the new season.     

Bronchial stump fistula :treatment with covered retrievable hinged metallic stents-preliminary clinical experience

Objective To evaluate the preliminaily clinical efficacy and retrievability of a retrievable hinged covered metallic stent in the treatment of the bronchial stump fistula （BSF）. Methods Between April 2003 and March 2005, 8 patients with bronchial stump fistula after pneumonectomy or lobectomy were treated with two types （A and B） of retrievable hinged covered metallic stents. Type A stent was placed in 6 patients and type B in 2 under fluoroscopic guidance. The stent was removed with a retrieval set when BSF was healed or complications occurred. Results Stent placement in the bronchial tree was technically successful in all patients, without procedure-related complications. Immediate closure of the BSF was achieved in all patients after the procedure. Stents were removed from all patients but one. Removal of the stents was difficult in two patients due to tissue hyperplasia. Patients were followed up for 6 - 21 months. Placement of the stents remained stable in all patients except one due to severe cough. Permanent closure of BSF was achieved in 7 （87.5%） of 8 patients. Conclusion Use of a retrievable hinged covered expandable metallic stent is a simple, safe, and effective procedure for closure of the BSF. Retrieval of the stent seems to be feasible. （J Intervent Radiol, 2007, 16： 253-257）