基因探针和PCR对鲤吉陶单极虫的检测

采用液氮冷冻研磨法破碎吉陶单极虫（Ｔｈｅｌｏｈａｎｅｌｕｓｋｉｔａｕｅｉ）孢子，按常规法提取其基因组ＤＮＡ。取ＤＮＡＥｃｏＲＩ消化产物，采用低熔点琼脂糖回收法制备大（３．０～１５．０ｋｂ）、小（０．５～３．０ｋｂ）片段，将其克隆于ｐＢｌｕｅｓｃｒｉｐｔｋｓＥｃｏＲＩ位点，经α－互补现象、酶切鉴定和虫体ＤＮＡ生物素标记探针筛选，构建了含２３个克隆的基因文库，克隆片段介于０．９～６．８ｋｂ之间；经阳性克隆标记筛选及特性分析，制备了长度为０．９ｋｂ（１９号质粒克隆片段）的探针，该探针具有较强的敏感性和特异性。对该探针两端ＤＮＡ序列进行分析，设计出１对引物。上游引物序列为：５′ＴＴＴＣＧＡＡＣＣＣＧＣＡＣＡＡＣＡＡＧ３′，下游引物序列为：５′ＴＧＡＧＴＧＧＡＴＡＧ－ＴＡＴＣＧＣＴＧＣ３′。应用该对引物对虫体进行了检测     

TaqMan探针荧光RT-PCR检测狂犬病病毒

根据GenBank已公布的狂犬病病毒(rabies virus,RV)核蛋白(N)基因序列设计并合成一对特异性的引物和探针,建立基于TaqMan探针的荧光RT-PCR检测狂犬病病毒方法。对狂犬病疫苗提取核酸后进行RT-PCR扩增,将目的条带切胶回收,克隆测序,重组质粒作为标准阳性对照。对建立的TaqMan探针荧光RT-PCR方法做灵敏度、特异性、重复性及稳定性试验。结果显示,该方法可以达到10拷贝/μL的灵敏度,可以将狂犬病病毒与犬瘟热病毒、犬细小病毒、犬腺病毒、犬冠状病毒和犬副流感病毒分开,方法重复性好,稳定可靠。     

H3亚型猪流感病毒荧光定量PCR检测方法的建立

通过RT-PCR方法克隆了H3亚型猪流感病毒HA基因一段靶序列,构建重组质粒作为标准阳性模板.根据GenBank中的H3亚型猪流感病毒HA基因保守序列设计了用于FQ-PCR的1对引物和1条TaqMan探针.通过条件优化,以10倍系列稀释的质粒为标准品进行荧光定量PCR扩增,并制作标准曲线,建立了检测H3亚型猪流感的荧光定量PCR方法.结果表明,该方法检测灵敏度可达1.0×100拷贝/μL,线性范围为109～100,达10个数量级;对起始浓度为1.0×109、1.0×108、1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为13.68,18.21和20.57;变异系数分别为0.31%、0.17%和0.12%,均小于5%,说明此方法具有良好的准确性和重现性.对阳性组织病料的检测表明,该方法的检测灵敏度高出常规PCR,与套式PCR具有相近的灵敏度.     

HRP直接标记属特异性基因探针检测沙门氏菌的研究

以活化辣根过氧化物酶复合物（ＨＲＰ—ＰＢＱ—ＰＥＩ＋—ＮＨ３＋）直接标记沙门氏菌属特异性ＤＮＡ探针ｐＬＳ２和ｐＬＳ３，探针与靶ＤＮＡ杂交后催化发光底物，经增强型化学发光反应（ＥＣＬ），用普通Ｘ光胶片自显影（ＣＰＤ）检测沙门氏菌。经狭缝杂交（Ｓｌｏｔｂｌｏｔ），该法标记探针均可检测到０．１ｐｇ的纯质粒ＤＮＡ及１０３个未经培养的鼠伤寒沙门氏菌。Ｄｏｔ—ｂｌｏｔ杂交结果证明，ＨＲＰ标记的探针仅与沙门氏菌属细菌杂交，而与试验的其他肠道非沙门氏菌不杂交。本研究表明，ＨＲＰ直接标记基因探针化学发光自显影法检测沙门氏菌，安全、快速、简便，且有高度的敏感性和特异性，是一种有较大应用前景的非放射性标记探针杂交检测方法。     

小反刍兽疫病毒及山羊痘病毒N-ITR基因二联实时荧光定量RT-PCR检测方法的建立

Peste des petits ruminants virus (PPRV) and goat pox virus (GTPV) are the causative agents of two kinds of goats’ diseases-peste des petits ruminants and goat pox which can cause disaster economic losses. In order to detect the two viruses simultaneously and quickly, two sets of primers and relative probes were designed based on the nucleoprotein (N) gene of PPRV and the inverted terminal repeat (ITR) segment of GTPV, respectively. In order to work together in the same reaction, the probes were labeled with different fluorescent materials 5′FAM-TAMRA3′and 5′JOE-Eclipse3′,respectively. Results showed that the duplex Real-time RT-PCR assay was identified to be specific for PPRV and GTPV only and specific fluorescent signal could be detected, but the related viruses including fowl pox virus（FPV）and canine distemper virus (CDV) had no specific fluorescent signal. Positive recombinant plasmids (PPRV pMD18-T-N and GTPV pMD18-T-ITR) were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe N-ITR was found to be highly specific and sensitive with a detection limit of 10² copies/μL cDNA and 10³ copies/μL DNA for PPRV and GTPV, respectively. Finally, the duplex Real-time RT-PCR assay for simultaneous detection of PPRV and GTPV was established preliminarily in the study.     

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.     

应用核酸斑点杂交法检测鹅细小病毒(GPV)

利用鹅细小病毒 (GPV)核酸探针 ,设计并建立了核酸斑点杂交法 ,用以检测 GPV核酸。该方法特异性与敏感性均较好 ,其敏感度可达 3pg。应用该方法对 4例病变典型的小鹅瘟患鹅组织器官进行检测 ,结果表明 ,患鹅脏器中 ,小肠、心、肝、胰、脾组织含毒量较高 ,其中小肠组织含毒量最高 ,而脑组织中含毒量较低。应用琼脂扩散试验验证了核酸斑点杂交法的检测结果。     

Premature centromere division (PCD) identified in a hucul mare with reproductive difficulties

A hucul mare with reproductive abnormalities was examined during karyotype analysis. The karyotype was analysed based on evaluation of 860 metaphase plates in chromosome preparations. The use of fluorescence in situ hybridization (FISH) with an X chromosome painting probe showed premature X chromosome separation in 9.5% cases of examined chromosome spreads. In this report, we present the first identify premature centromere division (PCD) as a possible cause of abnormal X chromosome morphology in horses and as a probable cause of reproductive difficulties.     

基因芯片及应用

基因芯片是基于核酸分子杂交原理，利用光导原位化学合成或液相合成自动化点样技术，将大量的寡核苷酸探针固定于玻片，硅片、尼龙膜等固相支持物上，与荧光或同位素标记的样品核酸杂交，通过计算机系统检测与分析，获得有关的生物信息，它广泛应用于人类基因组研究，现代医学研究，动植物研究和环境保护等方面。     

大米粉的猪回肠氨基酸真消化率测定

大米加工副产品大米粉长期以来被添加到米糠中利用，显著降低了其利用率。本试验用三头施以回直肠吻合术猪，采用单因子设计，以大米粉为半纯合日粮，无氮日粮由醋酸纤维素提供粗纤维来测定内源值，测定了大米粉的猪回肠末端氨基酸表观消化率和真消化率。大米粉的回肠末端氨基酸的表观消化率的平均值为76．20％，真消化率的平均值为85.86％。这些数据可为我国氨基酸消化率数据库提供参考，并且可作为以可消化氨基酸为基础配制日粮时的依据。