TORCH与优生：Ⅲ用聚合酶链反应结合地高辛分子杂交产前诊断弓形虫感染

Prenatal diagnosis of Toxoplasma gondii infection by polymerase chain reaction combined with molecular hybridization of digoxigenin

根据本实验室筛选出的弓形虫（ＺＳ２株）特异ＤＮＡ克隆片段的部分顺序分析的数据，设计并合成特异的寡核苷酸引物对，建立多聚酶链反应（ＰＣＲ）诊断弓形虫感染的方法。不同来源弓形虫株和阳性标本ＤＮＡ的ＰＣＲ产物经电泳检测，均出现特异的扩增片段。以地高辛标记的该ＰＣＲ产物中弓形虫特异顺序的寡核苷酸为探针，对扩增产物进行斑点杂交分析，该探针能与阳性病例的扩增产物杂交，而不与阴性病例的扩增产物杂交。用ＰＣＲ结合地高辛分子杂交方法对不良生育史孕妇进行产前诊断，３４例外周血白细胞ＤＮＡ检测，２例阳性，分别为出生水肿胎儿和死胎；７６例羊水细胞ＤＮＡ检测，３例阳性，其中２例出生为无脑儿；３０例绒毛ＤＮＡ检测，４例阳性，均为难免流产。ＰＣＲ产物结合地高辛分子杂交方法可测出少至１０ｆｇ（１０－１４ｇ）的弓形虫ＤＮＡ，本方法更加特异敏感。

Based on the partial sequences of the specific DNA cloned fragment from T.gondii(ZS2 strain)a specific primer of the loigonucleotide for the Toxoplasma gondii DNA sequence has been designed and synthesized in our laboratory.The method of the diagnosis for infection of T.gondii by polymerase chain reaction(PCR)has been established.A specific amplified band was shown in the PCR amplifying products by gel electrophorisis from DNAs form different strains of T.gondii and positive cases.Using digoxigenin labeled the oligonucleotide of T.gondii specific sequence from the PCR product as probe and dot blot assay, the results showed that the probe only hybridized to the specific amplified DNAbands of positive cases, but did not hybridize to the amplified DNA bands of positive cases,but did not hybridize to the amplified DNA products of negative cases.The prenatal dingnosis of T.gondil infection for pregnant women with previous abnormal birth histories was tested by PCR combined molecular hybridization of digoxigenin.Two of the thirty four cases,diagnosing the DNAs from the peripheral blood leukocytes,were positive.One was as hydrocephalus and the other as dead fetal at birth.The DNAs from the amniotic fluid cells of seventy six cases were diagnosed,three were positive.Two among the three cases were as anencephalus at birth.Four of the thirty cases,diagnosing the DNAs from the chorion tissues,were positive,all of which were as habitual abortion.As little as 10 fg(10-14g)of purified DNA from T.gondii can be detected by the PCR products combined molecular hybridization of digoxigenin,the method is more specific and sensitive.