猪囊尾蚴特异性DNA片段的分离及聚合酶链检测方法的建立

目的分离猪囊尾蚴特异性ＤＮＡ技术片段并建立猪囊尾蚴聚合酶链反应（ＰＣＲ）检测方法。方法应用随意引物聚合酶链反应（ＡＰＰＣＲ）技术分离猪囊尾蚴特异性ＤＮＡ片段，根据其核苷酸序列设计引物，建立ＰＣＲ方法。结果应用ＡＰＰＣＲ技术从不同患者囊尾蚴结节活检标本中扩增得到基本一致的ＤＮＡ带谱。经Ｓｏｕｔｈｅｒｎ印迹杂交证实随意扩增所得片段与猪囊尾蚴ＤＮＡ同源。对其中一个片段再次扩增并克隆测序。依据测序结果设计合成引物，分别以猪囊尾蚴、猪和人组织ＤＮＡ作模板，仅有前者ＰＣＲ阳性，并可检出低至１０ｐｇ的猪囊尾蚴ＤＮＡ。结论猪囊尾蚴ＤＮＡＰＣＲ检测方法的建立为猪囊尾蚴感染的临床诊断和病原流行病学调查提供了实验基础。

Isolation of a DNA fragment specific for cysticercus cellulosae and establishment of polymerase chain reaction for cysticercus cellulosae DNA detection

Objective To isolate a DNA fragment specific for cysticercus cellulosae and to establish a polymerase chain reaction (PCR) for cysticercus cellulosae DNA detection. Method A DNA fragment specific for cysticercus cellulosae was isolated with arbitrarily primed PCR technique. Based on its nucleotide sequence, primers were designed and PCR assay for cysticercus cellulosae was established. Results Mostly accordant profiles were amplified from different patients' biopsied samples of cysticercus cellulosae nodus. Southern blot hybridization showed that arbitrarily amplified fragments were homologous to cysticercus cellulosae DNA. A 500 bp fragment with high consistency and repetitiveness was reamplified, cloned and then sequenced. A pair of primers from the sequence were designed and synthesized. DNA samples from cysticercus cellulosae, pig, and human were amplified respectively, and only the samples from cysticercus cellulosae were detected to be positive. Amplification of continuously diluted samples of cysticercus cellulosae DNA showed that a minimum of 10pg DNA could be detected by means of this PCR method. Conclusion The establishment of PCR assay for cysticercus cellulosae DNA detection provides laboratory basis for clinical diagnosis and etiological investigation of this pathogen.