原位杂交检测微小病毒B19方法的建立及临床应用

摘要

Objective To establish In situ bybridization(ISH) technique to detect parvovirus B19 and to knowthe viral location in cardiac tissue of congenital heart diseases(CHD). Methbods ISH method was establisbed with ran-domly primed,degoxigenin labeled B19 DNA probe of 1 112 bp long corresponding to parvovirus B19 capsid protein VP1genome. Results Dot blot hybridization showed that B19 DNA was positive with the sensitivity of 0.1 pg/μl, whileCMV 、HSV、TOX DNA were all negative. 7 out of 66 CHD cardiac tissues were B19 DNA positive by using ISH, while in38 cases of control group were all negative. B19 DNA positive signal was found in the nucleus of cardiac cell. Conclu-sion We have established ISH to detect B19 DNA that has high sensitivity(0.1 pg/μl)and specificity and have foundthat B19 DNA is located the nucleus of cardiac cell.%目的　建立原位杂交(ISH)方法　检测人微小病毒B19,并确定其在先天性心脏病(CHD)心脏组织细胞的定位分布。方法　以B19特异的衣壳蛋白VPl DNA内1 112 bp片段为模板，采用随机引物标记探针法，建立了ISH检测B19 DNA。结果　通过斑点杂交定性分析显示B19 DNA呈阳性，CMV、HSV和TOXDNA均呈阴性；定量分析B19 DNA浓度于0.1 pg/μl以上呈阳性。在66例先天性心脏病心脏组织中，检测到B19 DNA阳性7例，并发现B19 DNA阳性信号主要定位于心肌细胞核内，38例对照组健康心肌中均阴性。结论　 建立的ISH检测先天性心脏病心脏组织B19的方法　有良好的特异性及敏感性，敏感性达0.1 pg/μl，并确定B19 DNA定位于心肌组织的心肌细胞核内。