用普通琼脂糖代替低熔点胶回收DNA片段

为了建立一种直接从普通琼脂糖凝胶中回收DNA片段的简便实用的方法,采用聚合酶链式反应扩增人P53基因外显子7、8和其间的内含子7序列,用普通琼脂糖凝胶电泳,直接从凝胶中切下产物带,用加热熔化法回收DNA;紫外比色法测定回收率;用测序法鉴定回收产物质量。并用QIAquick Spin纯化柱对照。结果表明,本法回收的产物质量明显优于用QIAquick Spin柱回收,本法回收的产物用于测序效果极佳,回收率达80％,用QIAquick Spin柱回收率不到20％,差异非常显著（P<0.01）。证明这种方法回收PCR产物质量可靠,能代替低熔点胶回收DNA,有较大的实用价值。 Abstract： In order to find a simple and efficient method to isolate single or double?strand DNA fragment amplified by polymerase chain reaction (PCR),we used PCR method to amplify exon 7,exon 8 and intron 7 of human P53 gene, electrophoresis to identify products,fusion and phenol-chlorofom extraction (FPC) to isolate specific DNA from agarose gel,ultraviolet colorimetry to deteminate collected rate,and direct sequencing to identify the quality of recollected DNA. A control test was also made by using QIAquick Spin Colum．The results showed that the quality of PCR products recollected by using FPC method was very good．When the recollected DNA was used in sequencing,no matter what was single or double-strand DNA,the sequence data was clear and even,with low noise．The recollected rate of using FPC,which was over 80 per cent, was higher than that of using colum (lessthan 20 per cent), there were statistical significances (P<0.01).In the control test, it had a little non-specific DNA in the collected products,and the sequencing experiment of using double-strand products was failure．All above mentioned suggested that general agarose gelis efficient in place of low melting-temperature for isolating DNA fragment．     

结核感染T细胞斑点试验对肺结核病的临床诊断价值研究

目的：评价结核感染T细胞斑点试验（T-SPOT．TB）对肺结核病的临床诊断价值。方法：选择2012年2月～8月湘雅医院呼吸科住院病人中92例可疑肺结核患者进行T—SPOT．TB检测、结核菌素试验（PPD试验）、结核抗体、血沉及影像学检查及病史收集。分析和比较T．SPOT．TB与传统结核诊断方法的阳性率、特异度、灵敏度并对其检测结果进行相关性分析。结果：92例患者中,48例被确诊为肺结核,其中41例T．SPOT．TB结果阳性,44例非肺结核患者中5例T．SPOT．TB结果阳性。T—SPOT．TB检测的敏感度为85．4％,特异度为88．6％。T-SPOT．TB检测在结核组的阳性检出率（85．4％）显著高于传统检查方法PPD（37．5％,P〈0．01）、结核抗体（16．7％,P〈0．01）、血沉（66．7％,P〈0．05）,在非结核病组中的特异性（88．6％）显著高于血沉（36．6％,P〈0．0J）。PPD与T-SPOT．TB联合可提高诊断结核的阳性率（89．6％）。T．SPOT．TB检测仅与PPD试验的结果存在显著性差异（P〈0．05）。结论：T-SPOT．TB诊断肺结核的敏感性及特异性较传统的PPD实验、结核抗体更高,具有重要的I临床应用价值。