目的 比较评价磁珠法、柱子吸附法和煮沸裂解法3种不同的核酸提取方法对淋病奈瑟菌（淋球菌）DNA提取效率的差异。 方法 对已确诊的淋球菌培养基挑取20个小菌落制成悬液，做1︰1～1︰100 000稀释度稀释，分别使用磁珠法、柱子吸附法和煮沸裂解法进行DNA提取，用实时荧光定量PCR对其提取效率进行评价。 结果 在不同浓度淋球菌的DNA提取中，以磁珠法的敏感性和重复性最高，而柱子吸附法和煮沸裂解法次之，＜1×105 copies /mL以下的拷贝数，3种DNA提取方法之间重复性和敏感性差异明显，磁珠法的提取效率最高，柱子吸附法次之，煮沸裂解法最差；＞1×105 copies/mL的细菌拷贝数的样本，3种方法差异随浓度增高不断缩小。 结论 磁珠法提取淋球菌DNA的效率、敏感性和重复性最高，在实际临床科研工作中，各实验室可根据自身实际情况选择不同的DNA提取方法。
【摘要】 目的 研究ΔNP63/TAP63在上皮性卵巢肿瘤组织中的表达及其与临床病理特征的关系。 方法 运用荧光定量聚合酶链反应方法检测2002年-2004年54例卵巢上皮性肿瘤中ΔNP63/TAP63的基因水平。 结果 33例卵巢上皮细胞癌组织中ΔNP63的表达高于21例良性上皮性肿瘤中组织。卵巢上皮细胞癌中的表达强度与肿瘤组织病理学分期相关（Plt;0.05），良性肿瘤的表达低于恶性肿瘤（Plt;0.05）。ΔNP63的表达高于TAP63（Plt;0.05）；各组间TAP63的表达差异无统计学意义（Pgt;0.05）。 结论 ΔNP63在上皮性卵巢癌中高表达，可能成为上皮性卵巢癌诊断及预后的分子标志物。【Abstract】 Objective To explore the expression of ΔNP63 / TAP63 in human epithelial ovarian tumor tissues and its relationship with the clinicopathological features. Methods Fluorescent quantitative PCR method was used to detect 54 cases of ΔNP63 / TAP63 gene level in 54 patients with epithelial ovarian tumors diagnosed between 2002 and 2004. Results ΔNP63 expression in the 33 cases of ovarian epithelial cell carcinoma was higher than that in the 21 cases of benign epithelial tumor tissue. The expression in ovarian epithelial cell carcinoma was concerned with the pathological staging of tumor (Plt;0.05); the expression in benign tumors was lower than that in the malignant tumors (Plt;0.05). In all cases, the expression of ΔNP63 was higher than that of TAP63 (Plt;0.05); the difference in the expression of TAP63 among the groups was not significant (Pgt;0.05). Conclusion ΔNP63 in epithelial ovarian cancer is highly expressed, which may become the molecular makers with diagnosis and prognosis of epithelial ovarian cancer diagnosis in the future.
ObjectiveTo explore the expression of growth associated protein-43 (GAP-43) in spasm segment and expansion segment of hirschsprung disease (HD), and to explore the pathogenesis of HD. MethodsThe expression of GAP-43 in 30 patients with HD who underwent surgical resection for absence of enteric plexuses from Jan. 2012 to Jun. 2013 in Shen zhen Children's Hospital were analyzed by using immunohistochemistry method and real-time PCR method. Aganglionic tissues of all patients were included as spasm group, and ganglionic tissues of the same patients were served as expansion group. Then comparison of the expression levels of GAP-43 mRNA and its protein between 2 groups was performed. Resultsof real-time RCR showed that the expression level of GAP-43 mRNA in expansion group was higher than that of spasm group (0.119 0 vs. 0.052 8, P<0.05). Immunohistochemistry results showed that GAP-43 protein expressed both in the myenteric plexus and ganglionic plexus of submucos in all patients, but lighter in spasm group. Compared with ganglionic plexus of circular muscle layer and longitudinal muscle layer/ganglionic plexus of submucosa in expansion group, the average optical density values at corresponding sites of intestinal tissues in spasm group were both lower (P<0.05). ConclusionExpression of GAP-43 protein is lower in spastic intestinal tissue of patients with HD, which suggests that down-regulation of GAP-43 protein may be a risk factor for HD.
ObjectiveTo establish a method that can eliminate the pollution of endogenous nucleic acid in the real-time quantitative polymerase chain reaction (PCR) reaction system, which can be used to reduce or eliminate the false positive rate of real-time PCR assay in detection of postoperative intracranial bacteria infection.MethodsAt first, eliminated the pollution of endogenous nucleic acid in the real-time PCR reaction system. Then, with mixed bacteria DNA as a template, multiple PCR was used to specifically identify the gram-negative bacteria. Meanwhile, evaluated the text line and sensitivity of the multiple PCR after eliminating pollution in detecting the DNA of the mixed bacteria.ResultsThe method established could quickly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and it didn’t affect the Taq enzyme activity and the amplification efficiency in PCR system, with the minimum detection limit of 102 CFU/mL (Staphylococcus aureus and Pseudomonas aeruginosa), which was the same to the culture method. The enzyme cutting method had no significant effect on the activity and amplification efficiency of the enzyme in PCR system, It had no effect on PCR reaction system and primer specificity (Ct=32, ΔRn=200). However, the filtration method significantly reduced the PCR amplification efficiency (Ct=32, ΔRn=150).ConclusionsThis method can easily and rapidly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and greatly reduce the false positive of PCR detection. It is able to timely and accurately diagnose the intracranial bacteria infection, which is significant for clinical testing.