聚合酶链反应技术检测弓形虫核酸的研究

本文通过克隆的ＲＨ株弓形虫核酸ＴＧＲ４片段，设计并合成了一对长度为２０ｂｐ的寡核苷酸引物，扩增靶序列长度为７７８ｂｐ，建立了聚合酶链反应技术检测弓形虫核酸的特异敏感方法。检测７株弓形虫株及临床疑似弓形虫病患者样本，均出现特异扩增带，而其它８种病原体以及正常人及动物的ＤＮＡ均未见特异扩增带。扩增产物用地高辛素标记作探针，与以上出现特异扩增带的ＤＮＡ模板能斑点杂交，而与无扩增带的其余模板ＤＮＡ无斑点杂交。结果显示，该引物具有高度的保守性及特异性。在含１０５个人白细胞中，可检测到２个弓形虫ＤＮＡ或１ｐｇＤＮＡ。并通过人工感染弓形虫鼠血样品的检测表明，所建立的聚合酶链反应体系具有高度的敏感性，能早期检出弓形虫感染样品，在弓形虫病的临床诊断中具有重要的应用价值。     

HPV6，11型在女性生殖器尖锐湿疣和乳头瘤样增生中的分布

从３４２名上海地区患者的外阴、阴道和宫颈的病变部，采集４８７份活检标本。５种病理诊断类型，经斑点杂交法检测，ＨＰＶ６、１１ＤＮＡ总阳性率：尖锐湿疣８５％、乳头瘤样增生１０．１０％、慢性宫颈炎７．１４％、鳞状上皮增生５％、生殖器正常粘膜２．６５％。５１例尖锐湿疣和１０例乳头瘤样增生，ＨＰＶ６、１１和６＋１１感染阳性率分别为：１５．６９％、２５．４９％、５８．８２％和２０％、３０％、５０％。Ｓｏｕｔｈｅｒｎ印迹转移杂交与斑点杂交的符合率，在尖锐湿疣中，ＨＰＶ６、１１和６＋１１分别为７５％、９２．３％、８６．７％；在乳头瘤样增生中，分别为１００％、３３．３％、６０％。ＡＢＣ法检测阳性率仅为６１．６７％和０％。本调查结果证实，上海地区女性尖锐湿疣和乳头瘤样增生与ＨＰＶ６、１１型感染密切相关，且以ＨＰＶ两种型别（ＨＰＶ６＋１１）混合感染为主。     

Molecular characterization and cloning of sheep mitochondrial DNA

Summary Mitochondrial DNA from the liver of a single Rasa Aragonesa sheep has been isolated and characterized. The size of the genome, determined by restriction enzyme analysis, was found to be 16.58 kbp. The cleavage sites for the restriction endonucleases BamHI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI were mapped, and the gene organization deduced through heterologous hybridization using different cloned fragments of the rat mitochondrial genome. Fragments representative of the entire sheep genome were cloned in plasmid vectors pGEM3Z and pUN121.     

聚合酶链反应标记输血传播病毒(TTV)地高辛素探针的初步应用

用聚合酶链反应法（ＰＣＲ）制备地高辛素标记的输血传播病毒（ＴＴＶ）探针，并与巢式ＰＣＲ法（ｎＰＣＲ）比较。结果该探针具有ＴＴＶ特异性，其灵敏度为１０ｐｇＤＮＡ。应用该法检测１０８份非甲～庚型肝炎病人的血清标本，ＴＴＶＤＮＡ阳性率为１８５％（２０／１０８）；检测２２份病人的粪便标本，ＴＴＶＤＮＡ阳性率为２７３％（６／２２）。该法与ｎＰＣＲ法的总符合率为９６９％（１２６／１３０）。结论：用ＰＣＲ法直接制备地高辛素标记ＤＮＡ探针简便、快速、灵敏度高，特异性好。应用该法从６名病人的粪便中检出ＴＴＶＤＮＡ，提示ＴＴＶ有可能通过粪口途径传播。     

非放射性探针杂交检测早期钩端螺旋体病患者血清钩体DNA的研究

作者首次应用非放射性地高辛配基、光敏生物素探针并以~(32)P探针作对照,斑点杂交检测33创早期钩体病人血清。结果,三种探针杂交的阳性率分别为69.70％(23/33)、27.27％(9/33)、57.58％(19/33)。地高辛配基探针的杂交阳性率高于血培养(20/33),明显优于光敏生物素探针(p<0.01),稍高于~(32)P探针。阴性对照标本中,光敏生物素探针杂交1例出现阳性,地高辛配基,~(32)P探针无阳性结果出现。     

地高辛素、生物素标记产毒性大肠杆菌 Ⅰ型不耐热肠毒素ADN探针的制备及应用

采用随机引物法制备地高辛素、生物素和~(32)P标记产毒性大肠杆菌Ⅰ型不耐热肠毒素(LT_1)DNA探针。地高辛素、生物素和~(32)P LT_1探针分别在DNA斑点杂交试验中检测到0.1pg、1pg和0.1pg的质粒DNA,在菌落原位杂交试验中检测到12、120和12个细菌/ml,在粪便斑点杂交试验中检测到24、240和24个细菌/ml。采用RNA酶A和蛋白酶K消化处理滤膜,消除了假阳性,使得地高辛素、生物素LT_1探针的特异性与~(32)PLT_1探针完全一致。检测143株自腹泻病人分离的大肠杆菌,14株(9.79％)阳性,3种探针结果相同。     

Expression of AMPA receptors in rat superior colliculus and effect of orbital enucleation

We analyzed the distribution and the morphological characteristics of neurons expressing AMPA-type glutamate receptor subunits (GluR1 and GluR2) in the superficial partition (stratum zonale (SZ), stratum griseum superficiale (SGS) and stratum opticum (SO)) of the rat superior colliculus. GluR1-expressing neurons had round or ovoid somata in SGS and round or fusiform somata and primary dendrites extending tangential or horizontal side in SO. On the other hand, GluR2-expressing neurons mainly corresponded to vertical fusiform cells with vertically oriented dendrites in SGS and medium-sized stellate or ovoid cells with many primary dendrites in SO. The results suggest that the expressions of GluR1 and GluR2 are differentially regulated in individual neurons of the superficial partition. To analyze the effect of retinal deafferentation on the expression of the GluRs, we performed unilateral orbital enucleations in rats within a week after birth. Thirty days after retinal lesioning, lower expression of GluR2 mRNA was observed in the neurons of the contralateral side as compared with that of the ipsilateral side in SO, but not in SGS. These results indicate that GluR2 expression in the SO neurons is regulated by the correct afferentation from the retina.     

Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou

Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1-128 and 2-256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17beta (E2). The changes in serum levels of Chgs and Vg in male fish differed according to the E2 dose. When fish were given a 5 mg/kg body weight (BW) of E2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 microg/kg BW of E2 injection. A similar trend was seen in the response time to exogenous E2. Serum Chgs were induced from 8h after E2 injection and reached a peak of about 5 microg/ml at 24h. Although Vg was not detected until 8h after E2 injection, its levels remained considerably low at around 0.03 microg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 microg/kg BW of estrogen: the long-term exposure of fish to E2 showed that Chg H could be induced from a lower dose of E2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E2.