地高辛素标记的DNA探针快速鉴定产不耐热肠毒素大肠杆菌的研究

用地高辛素标记的６７０ｂＰ－ＬＴ－ＤＮＡ片段作为探针，用菌落原位杂交法对９株标准参考株进行检测，结果全部相符。对７０株从临床分离的菌株也进行菌落原位杂交．并与３２－Ｐ标记的探针进行比较．结果地高辛素探针的特异性为９７．７％，敏感性为１００％，符合率为９８．６％。从含正常肠道菌的粪便中可以直接检出ＬＴ－ＥＴＥＣ而无须经纯培养。定量试验表明本法最低检出量为６ＣｆｕＬＴ－ＥＴＥＣ／斑点。整个检测过程短．仅须２４～３０ｈ。探针的稳定性好，有效期可长达１年。     

The use of DIG-labelled cRNA probes for the detection of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) in orchids

DIG-labelled sense and antisense cRNA probes were synthesized from cDNA clones of CymMV and ORSV for virus detection in infected plants. A slot-blot hybridization assay was developed using either crude leaf extracts or total RNA from infected leaves. The assay could detect 50 and 250 pg of purified CymMV and ORSV RNA, respectively. As little as 30 mg of Nicotiana benthamiana infected leaves was sufficient to provide positive detection. CymMV and ORSV were detected at 3125 and 625 times dilution of leaf extracts, respectively. The DIG-labelled cRNA probes are stable for more than a year. This method is sensitive, reliable and suitable for large-scale routine testing of plant viruses. By using the two DIG-labelled cRNA probes in situ, CymMV and ORSV were localized in systemically infected leaves and stems of N. benthamiana and orchids.     

聚合酶链反应标记轮状病毒地高辛素探针的初步应用

目的为探讨聚合酶链反应（ＰＣＲ）技术标记的地高辛素（ＤＩＧ）探针的特异性和敏感性。方法用聚合酶链反应技术制备地高辛素标记的人类轮状病毒（ＨＲＶ）ｃＤＮＡ探针，经ｃＤＮＡ－ＲＮＡ斑点杂交。结果该探针具ＨＲＶ特异性，可检出１０ｐｇ的ＲＮＡ。应用该项技术检测了１２０份婴幼儿腹泻粪样标本，其阳性率为６５％，明显高于ＰＡＧＥ方法（４９．１％）的阳性率。结论ＰＣＲ方法直接制备地高辛素标记的ｃＤＮＡ探针方便、快速、特异性好、标记率高。     

地高辛标记RT-PCR检测甲肝减毒活疫苗的研究

以编码甲肝病毒ＶＰ３ 羧基端的基因区为目标区,运用地高辛标记ＲＴＰＣＲ 法对甲肝疫苗进行检测,并与ＴＣＩＤ５０ 检测法进行比较。建立甲肝减毒活疫苗滴度的地高辛标记ＲＴＰＣＲ 检测法。结果显示：地高辛ＲＴＰＣＲ 法灵敏度略高于ＴＣＩＤ５０ 法,特异性与ＴＣＩＤ５０ 相似,且快速简便,易于推广应用。结果表明：地高辛标记ＲＴＰＣＲ 法是一种简便、快速、灵敏的甲肝病毒检测方法,可用作疫苗的常规检测。     

神经生长因子高亲和力受体互补核糖核酸探针的研究

目的 制备用地高辛标记的神经生长因子高亲和力受体(Trk A)正、反义互补核糖核酸(cRNA)探针的研究。方法 设计Trk A引物，构建pGEM-T Easy-Trk A重组质粒，分别用ApaI和SacI进行酶切得到线性DNA片段，以SP6和T7RNA聚合酶转录合成带有地高辛标记的高比活度的正、反义单链cRNA探针。结果 经斑点杂交证实，该探针敏感性高、特异性强。Trk A-ApaⅠ探针稀释12500倍后的浓度为40ng／μl；Trk A-SacⅠ稀释1：2500倍后的浓度为8ng/μl。结论 成功制备了地高辛标记的Trk A cRNA探针，为毒理学中有关Trk A mRNA在组织、细胞的表达和分布的研究提供了有效工具。     

非放射性乙型肝炎病毒核酸探针的制备和应用

非放射性的生物素（Ｂｉｏｔｉｎ）和地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ）标记的乙肝病原基因探针（Ｂｉｏ－ＨＢＶＤＮＡ和Ｄｉｇ－ＨＢＶＤＮＡ探针），特异性强，灵敏度高，最低检出值分别为１ｐｇ和０．５ｐｇ。３２０份血清检测了ＨＢＶＤＮＡ和ＨＢｅＡｇ，并与α－_（３２）Ｐ标记的探针检测结果相比较，它们的特异性分别为９６．５％、９８．１％和８８．９％；灵敏度分别为８１．７％、８６．７％和６１．７％；符合率则为９３．８％、９５．９％和８３．８％。应用Ｄｉｇ－ＨＢＶＤＮＡ探针，作斑点杂交试验，在乙肝患者血清、白细胞、尿、粪、唾液以及旅客列车的桌面、车箱门把手、厕所门把手和水笼头上，均可检得阳性结果。我所研制的非放射性乙型肝炎病原基因诊断试剂，为科学研究和临床检测提供了极为有用的工具。     

Fluorescence in situ hybridization (FISH): Detection of biotin- and digoxigenin-labeled signals on chromosomes

Summary A laboratory protocol of fluorescence in situ hybridization (FISH) on the air-dry chromosome preparation is described. This protocol contains procedures for preparing solutions; probe-labelings with biotin and digoxigenin by means of nick translation, random priming, and polymerase chain reaction; probe-annealing; and signal detection. Uses of solutions are simplified; compositions of each solution are outlined; and, whenever possible, easily accessible materials and supplies are used so that the FISH technique can be applied by all cytogenetic laboratories. Annealed signals at 4–5 kb or over were detectable, and background signals, if any at all, were low. The slides processed for FISH can be used subsequently for conventional chromosome bandings. Therefore, the same slide can be used for both conventional and molecular karyotype studies.     

癌转移抑制基因nm23在人肺癌中的转录表达研究

目的　探讨nm23基因在人肺癌发生、发展中的作用。方法　采用狭缝印迹杂交技术和非放射性地高辛标记检测系统,检测了143份不同部位、不同性质人肺组织中的nm23H1和nm23H2基因的mRNA表达。结果　nm23H1和nm23H2基因的mRNA表达均有从正常肺组织、肺良性病变组织、非癌肺组织、癌灶组织到癌转移淋巴结组织逐渐降低的趋势。其中,肺癌组织的nm23H2基因mRNA表达较正常肺组织显著较低(P<0.05),癌转移淋巴结组织的nm23H1和nm23H2基因mRNA表达均较正常肺组织显著降低(P<0.05)。肺癌组织的nm23基因表达与淋巴结有无癌转移无明显关系(P>0.05)。结论　nm23基因表达降低可能与肺癌发生有关,未发现nm23基因有癌转移抑制作用     

chOPG RNA探针的制备及其在鸡骨组织的原位杂交

目的 制备地高辛标记的禽OPG cRNA探针.方法 将目的片段重组到T载体中,构建chOPG/pGM-Teasy重组质粒,EcORI酶切鉴定并测序验证.分别用SalI和NcoI进行酶切得到线性化DNA模板,用Sp6和T7 RNA聚合酶体外转录合成地高辛标记的正反义RNA探针.运用斑点杂交的方法检验探针的标记效率,并通过骨组织原位杂交反应进一步检测探针标记是否制备成功.结果 成功构建出chOPG/pGM-Teasy质粒.结论 获得的高效正、反义chOPGRNA探针,为进一步研究OPG在蛋鸡各组织中的表达奠定基础.     

In situ PCR for in vivo detection of foreign genes transferred into rat brain

Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320–330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, β-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82°C before adding Taq polymerase (‘hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.