不同切口白内障超声乳化摘除术对泪膜的影响

目的：观察巩膜隧道切口和透明角膜切口对白内障超声乳化摘除术患者术后泪膜的影响。方法临床纳入63例患者共70眼行白内障超声乳化吸除联合人工晶体植入术的非干眼患者,根据切口不同分为巩膜隧道切口组（32例,35眼）与透明角膜切口组（31例,35眼）,两组患者均进行折叠人工晶状体植入。术后第1、7、14、30、90天观察两组患者泪液分泌试验（SIt）、泪膜破裂时间（BUT）以及自觉干眼感觉等。结果术后第1天及第7天,两组患者自觉干眼症状明显,巩膜隧道切口组干眼症状评分[（2.55±0.96）、（1.49±0.62）分]明显高于透明角膜切口组[（1.18±0.72）、（0.98±0.71）分],差异有统计学意义（P〈0.05）;SIt试验显示,术后第1天,巩膜隧道切口组与透明角膜切口组患者SIt分别为（19.55±2.47）、（17.62±2.51）mm,均较术前显著升高,巩膜隧道切口组长于透明角膜切口组,差异有统计学意义（P〈0.05）;BUT试验显示,术后第1、7、14天,两组BUT均较术前显著缩短,巩膜隧道切口组[（4.07±0.63）、（4.62±0.86）、（6.37±0.67）s]长于透明角膜切口组[（3.42±0.67）、（4.06±0.90）、（5.88±0.77）s],差异有统计学意义（P〈0.05）。结论术后早期,进行透明角膜切口白内障超声乳化摘除术的患者,对泪膜的影响较小,但临床干眼症状要重于进行巩膜隧道切口的患者。术后晚期,两种切口进行白内障超声乳化摘除术对泪膜无明显影响。     

多重荧光PCR-熔解曲线法同时检测常见病原真菌方法学的建立及诊断价值

目的建立多重荧光PCR-熔解曲线法，并评价其同时检测假丝酵母菌、曲霉菌及新型隐球菌的诊断价值。 方法建立多重荧光PCR-熔解曲线法并对其检测体系进行优化。收集临床高度怀疑为侵袭性真菌感染（IFI）的各类临床样本179例，其中血液65例、深部痰液35例、尿液30例、脑脊液18例、胸腹水12例、肺泡灌洗液10例、新鲜肺组织9例。通过敏感性、特异性、重复性实验验证多重荧光PCR-熔解曲线法的检测性能，并应用受试者工作特征（ROC）曲线评价该方法的诊断效能。 结果建立的多重荧光PCR-熔解曲线法可同时检测8种常见病原真菌，包括4种假丝酵母菌（白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌、克柔假丝酵母菌）、3种曲霉菌（烟曲霉、黄曲霉、黑曲霉）和新型隐球菌。其最低检测限为1 × 10⁴ cfu/mL，且常见细菌无扩增反应，重复性实验解链温度波动小于0.5 ℃。179例临床样本中，以培养法、镜检法、病理诊断等"金标准"方法诊断IFI阳性为112例，多重荧光PCR-熔解曲线法检测阳性为96例。多重荧光PCR-熔解曲线法同时检测假丝酵母菌、曲霉菌及新型隐球菌的敏感度为0.857，特异度为0.970，阳性预测值为0.980，阴性预测值为0.802，ROC曲线下面积为0.914，95%置信区间为0.868 ~ 0.959，P < 0.001。 结论本研究建立的多重荧光PCR-熔解曲线法可快速、准确、高通量同时检测假丝酵母菌、曲霉菌及新型隐球菌，对于IFI的早期诊断具有重要意义。     

Oral gram-positive bacterial DNA-based identification of saliva from highly degraded samples

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40–80 °C in wet conditions; microbial decomposition: 1–5 days in humid soil; and ultraviolet (UV) irradiation: 0.01–1 J/cm²). We compared saliva markers’ detectability from the degraded samples—oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1–5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose–dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2–5 days) and UV (0.1–1 J/cm²) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.     

荧光杂交探针实时聚合酶链式反应快速检测甘露聚糖结合凝集素启动子区基因变异方法的建立

目的建立用荧光杂交探针实时聚合酶链式反应（Real-timePCR）检测人甘露聚糖结合凝集素（MBL）启动子区基因变异新型的基因分型法——Tm基因分型法。方法收集30例健康献血员外周抗凝全血,提取基因组DNA。通过LightCycler实时PCR仪描绘扩增的目标DNA片段的熔解曲线,根据熔解温度（Tm）峰值判定待测标本的基因变异类型。最后,用基因测序法检测PCR产物来验证Tm基因分型法的准确性。结果建立了新型的Tm基因分型法,且测定PCR产物的时间仅180min,检测结果与测序法完全吻合。结论Tm基因分型法检测人MBL启动子区基因变异快速、准确,重复性好,具有广泛的临床应用前景。     

熔解曲线分析法用于HPA-15基因分型的研究

目的探讨熔解曲线分析法用于HPA-15基因分型,了解大连地区汉族人群HPA-15基因多态性。方法设计合成HPA-15等位基因特异性引物和1条公共引物,其中在2条等位基因特异性引物的5'末端分别加入具有不同长度并富含GC碱基的序列。使用这3条引物,进行实时PCR反应。在PCR反应后,进行熔解曲线分析,依据PCR产物熔解温度的差异,进行基因分型。应用熔解曲线分析法和常规PCR-SSP法对100名大连地区汉族人群的血液标本进行HPA-15基因分型。此外对熔解曲线分析法的重复性进行评价。结果 100个血液标本的熔解曲线分析法和常规PCR-SSP法的基因分型结果是完全一致的。10个血标本的2次熔解曲线分析结果是一致的。大连地区汉族人群HPA-15a、-15b基因频率分别为0.58和0.42。大连地区汉族人群与国内部分地区汉族人群相比,HPA-15等位基因频率的差异无统计学意义(P>0.05)。与新疆维吾尔族人群相比,HPA-15等位基因频率差异有统计学意义(P<0.05)。结论 HPA的熔解曲线基因分型方法具有快速、简单、准确、通量高、重复性好等优点,要优于常规的PCR-SSP法。在国内HPA-15等位基因分布存在着种族差异。     

巩膜扣带术治疗多发性裂孔性视网膜脱离应用硅胶与硅海绵垫压效果观察

目的探讨联合应用硅胶与硅海绵垫压的巩膜扣带术治疗多发性裂孔性视网膜脱离的临床疗效。方法多发性裂孔性视网膜脱离31例(31眼)在我院行联合应用硅胶及硅海绵垫压的巩膜扣带术,术中采用硅海绵垫压较大的和(或)较多的裂孔区,用硅胶垫压较小的和(或)较少的裂孔区。结果术后随访12～30个月,31只眼中,手术一次性视网膜完全复位29眼(93.54%),2例再次行玻璃体切割术后网膜复位;矫正视力在0.1以上者14眼;所有患者未见严重并发症。结论联合硅胶与硅海绵垫压手术是治疗多个裂孔视网膜脱离的有效治疗方法。     

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid–-based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02?×?10^?1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.     

乙型肝炎病毒全长基因组的扩增

目的:探讨扩增乙肝病毒全长基因组合适的PCR反应条件。方法:设计针对负链5′末端引物,PCR一步法扩增乙肝病毒全长基因。改变PCR反应条件,决定最佳退火温度和最佳引物浓度,并观察不同乙肝病毒DNA模板量对PCR扩增效率的影响。结果:最佳退火温度为68℃,最佳引物浓度为0.5μmol/L,模板量在150拷贝以上能扩增出乙肝病毒全长基因,但模板量达到105拷贝抑制扩增效率。结论:退火温度为68℃、引物浓度为0.5μmol/L、乙肝病毒DNA模板量在150～105拷贝为乙肝病毒全长基因扩增的合适PCR反应条件。