微小隐孢子虫种特异性基因片段的克隆及诊断引物的确定

目的 寻找用于诊断微小隐孢子虫病的特异性引物。 方法 在RAPD分析过程中获得了 1条微小隐孢子虫 [Cryptosporidium parvum (C .parvum ) ]种特有 712bp的基因片段 ,将该片段分离、纯化、克隆和测序 ,据此序列合成一对特异性引物FF。用引物FF扩增美国 2株C .parvum和中国 4株C .parvum ,并用该引物与Morgan( 1996 )发表的C .parvum种特异性诊断引物 0 2 1作对照 ,检测镜检C .parvum卵囊阴性的兔粪样 35份和人粪样 5 5份。 结果 引物FF扩增 6株C .parvum均能产生预计 6 0 3bp的片段 ,引物FF和引物 0 2 1粪样检测结果完全一致 ,其敏感性是可检出 1个卵囊的DNA。 结论 获得的引物FF特异性强 ,敏感性高 ,可用于微小隐孢子虫病的诊断。

诊断引物应用于我国三种重要赤眼蜂分子鉴定的研究

本研究根据螟黄赤眼蜂rDNA_ITS2序列设计了螟黄赤眼蜂TrichogrammachilonisIshii特异引物 ,同时采用文献中发表的松毛虫赤眼蜂TrichogrammadendrolimiMatsumura和玉米螟赤眼蜂TrichogrammaostriniaePangetChen的特异引物以及赤眼蜂属Trichogramma特异引物对赤眼蜂成虫和寄主卵样品进行了PCR特异扩增分析。结果表明 ,采用上述特异引物可从单头蜂稳定地扩增出明显的目的DNA条带 ,并且分子鉴定结果与形态学鉴定结果完全吻合。因此 ,采用上述 3对特异引物可以实现对我国 3种重要赤眼蜂种 ,即松毛虫赤眼蜂、螟黄赤眼蜂和玉米螟赤眼蜂的分子鉴定。

全基因组扩增在DMD植入前遗传学诊断中的可行性研究

目的:建立单细胞扩增前引物延伸法及巢式PCR技术,研究该技术在杜氏肌营养不良症植入前遗传学诊断中的可行性。方法:获取单个正常男女淋巴细胞和无杜氏肌营养不良(DMD)家族史的单个卵裂球,15碱基随机引物PEP扩增单细胞全基因组,再以巢式PCR技术检测PEP反应产物中DMD基因外显子8,17,19,44,45,48及Y染色体上的睾丸决定基因SRY。结果:单个淋巴细胞和单个卵裂球的6个DMD外显子扩增成功率分别为97.2％(175/180)和100％(60/60),单个男性淋巴细胞SRY的扩增成功率为100％(15/15),而单个女性淋巴细胞无SRY的扩增(0/15)。结论:检测单细胞DMD基因外显子8,17,19,44,45,48和SRY的PEP-巢式PCR技术具有较高扩增成功率和特异性,可望应用于 DMD的植入前遗传学诊断的单细胞基因诊断中。

PCR BASED TECHNIQUE FOR IDENTIFICATION AND DETECTION OF TRICHOGRAMMA SPP. (HYMENOPTERA: TRICHOGRAMMATIDAE) WITH SPECIFIC PRIMERS

The rDNA ITS2 regions of T. dendrolimi Matsumura and T. ostriniae Pang et Chen (Hymenoptera: Trichogrammatidae) were cloned and sequenced. The homologous sequences available in GenBank were retrieved and analyzed, and then specific primers were designed for molecular identification and detection of T. dendrolimi. Repeated screening showed that PCR amplification by the diagnostic primers enabled the differentiation of not only bulk samples and single adult (male or female), but also eggs and juveniles, which was not possible by conventional methods. The advantage of this system over morphology based systems is that non specialists are able to identify individuals or trace specimens efficiently. The derived molecular detection technique was then used to identify 12 specimens collected from different localities on the Chinese mainland; the results showed that this protocol could be applied to molecular monitoring of Trichogramma species in the field.Finally,ITS2s of 6 geographical populations of T.dendrolimi (TdCHA,TDJL,TdXZ,TdRH,TdGZ and TdYBL) were cloned and sequenced.The multialignment analysis of intraspecific ITS2 se quences showed that the diagnostic primers have their own theoretical bases.