鉴别牛早期胚胎性别PCR方法引物的设计与筛选

根据牛Y-染色体特异重复序列、睾丸特异蛋白基因以及性别决定基因序列设计合成5对公牛Y-染色体特异引物，依据牛骨胳肌α肌动蛋白前体基因和微卫星DNA序列设计合成4对牛DNA特异引物(内标引物)。单重PcR扩增牛基因组DNA，筛选出4对牛Y-染色体特异引物和1对牛DNA特异内标引物。将不同的Y-染色体特异引物与内标引物组合，多重PCR扩增牛基因组DNA、已知性别的牛成纤维细胞和克隆胚胎，筛选出2个可用于牛早期胚胎性别鉴别的PCR引物组合：B34／A12和B78／A12。     

兔早期胚胎PCR性别鉴定体系的建立

根据兔SRY基因序列设计两对引物作为兔雄性特异性引物,根据兔APP基因序列设计1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。     

牛胚胎性别鉴定荧光定量PCR方法的优化与应用

旨在建立一种可检测牛早期胚胎性别的复合探针体系,减少常规PCR方法电泳所带来的污染,提高鉴定准确率,降低鉴定成本。本研究以SRY为牛胚胎性别鉴定的目标基因,以进口YCD-PCR性别鉴定试剂盒为对照组（n=9 543）,荧光定量PCR的单引物分别扩增体系（荧光单扩,FSPSA,n=6 570）和双引物混合扩增体系（荧光双扩,FDPMA,n=22 238）分别做试验组,从性别鉴定效率、无反应率、雌雄胚胎百分率和比值、移植妊娠率、雌性胚胎母犊率、不同细胞取样数下的性别鉴定结果和鉴定成本等方面进行各组间比较。结果表明,荧光单扩组的雌性胚胎百分率和性别鉴定效率极显著高于其他两组（P<0.01）,无反应率极显著低于其他两组（P<0.01）,雌雄胚胎比值与其他两组差异较大（1.03∶1 vs 1∶1.03和1∶1.02）,雌性胚胎母犊率极显著低于荧光双扩组和对照组（P<0.01）;荧光双扩组的雌性胚胎百分率、雄性胚胎百分率和雌性胚胎母犊率均与对照组无显著差异（P>0.05）,雌雄胚胎比值与对照组相似（1∶1.03和1∶1.02）,无反应率极显著低于对照组（P<0.01）,性别鉴定效率极显著高于对照组（P<0.01）。取样细胞4~6组和7~10组的性别鉴定效率极显著高于1~3组和SD（separated & died cells）组（P<0.01）,而1~3组和SD组之间及4~6组和7~10组之间差异不显著（P>0.05）。移植妊娠率4~6细胞组最高（49.68%）,但各取样组间无显著差异（P>0.05）。荧光双扩法与对照组相比,鉴定成本下降了46.76%。结果显示,荧光双扩方法产母犊准确率最高,鉴定成本较低,取样4~6细胞时的移植妊娠率最高,是一种更可行的牛胚胎性别鉴定技术,更有利于产业化推广和应用。     

PCR扩增ZFY,ZFX序列鉴定奶牛性别的灵敏度研究

从克隆、测序所得奶牛ＺＦＹ、ＺＦＸ序列中设计１对高特异性引物（或探针），即分别特异于牛ＺＦＹ、ＺＦＸ的ＺＦ３、ＺＦ４，与克隆时使用的引物ＺＦ２搭配成ＺＦ２、ＺＦ３和ＺＦ２、ＺＦ４。利用这２对引物和ＺＦ１、ＺＦ２对奶牛基因组ＤＮＡ进行ＮｅｓｔＰＣＲ来判断性别，结果达到用超微量（８ｐｇ）ＤＮＡ，即可检出ＺＦＹ、ＺＦＸ序列而判定雌雄的灵敏度；用非同位素标记试剂盒（ＤＩＧ－Ｓｙｓｔｅｍ）标记ＺＦ３、ＺＦ４作为探针，对ＺＦ１、ＺＦ２扩增产物点杂交来判断性别，也达到同样的灵敏度     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

PCR法鉴别牛早期胚胎性别的研究进展

对PCR法鉴别动物胚胎性别的机理进行了分析,综述了近10年来相关操作技术的进展,包括早期胚胎取样、胚胎细胞DNA的提取、引物的设计、扩增条件的优化、扩增产物的检测和性控胚胎的冷冻等,并提出PCR法鉴别牛早期胚胎性别的前景与展望。     

利用多重PCR技术鉴别牛早期胚胎性别研究

本研究利用一对根据牛Y-染色体特异重复序列设计的牛Y-染色体特异性引物和一对根据牛微卫星DNA设计的牛常染色体内标引物,通过对PCR反应体系中Mg^2＋浓度和PCR反应条件中退火温度的优化,建立了牛早期胚胎性别鉴定的多重PCR反应体系。     

多重PCR技术同时检测牛传染性鼻气管炎和赤羽病方法的建立和应用

为满足同时对IBRV和AKAV进行快速诊断的需要,根据牛疱疹病毒1型(BHV 1)gB基因序列和赤羽病病毒(AKAV)的S基因序列,设计了2对针对这2种病毒的特异引物,并建立了多重PCR方法,分别对其最佳反应条件、特异性及敏感性进行了测定,结果表明:该方法能同时扩增得到2条与试验设计相符的311 bp(I BRV)和392 bp(AKAV)特异性条带;IBRV的灵敏度为0.13 pg/25μL,AKAV的灵敏度为1.04 pg/25μL。本试验方法的建立对于加强进出口牛IBRV和AKAV的检验检疫具有十分重要的意义。     

PCR方法鉴定牛早期胚胎性别研究进展

对用于早期胚胎性别鉴定的多重PCR、巢式PCR、引物一扩展预扩增PCR、非电泳PCR和LAMP法等扩增技术进行了综述,并针对早期胚胎性别鉴定技术在养牛业生产中的应用现状和存在问题进行分析与讨论,展望了PCR性别鉴定技术在养牛业中广阔的商业应用前景。     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

畜禽源3种革兰氏阴性杆菌多重PCR检测方法的建立与应用

为建立一种可准确、快速鉴定畜禽临床病例中3种常见革兰氏阴性杆菌的多重PCR检测方法,本试验针对大肠埃希菌23S rRNA基因、巴氏杆菌KMT基因和沙门氏菌invA基因分别设计合成了1对特异性引物,构建可同时快速鉴别大肠埃希菌、巴氏杆菌和沙门氏菌的多重PCR反应体系,并进行反应条件优化及方法性能评估。结果显示,所建立的方法最佳引物浓度分别为1.0、1.5和1.0 μmol/L,最佳退火温度为56 ℃。性能评价结果显示,所建立的多重PCR检测方法具有较好的特异性和敏感性,其中对沙门氏菌检测敏感性最高,为1.44 pg/μL。对70株革兰氏阴性临床分离细菌进行检测,结果表明,所建立的多重PCR检测方法能实现3种临床分离细菌的快速准确鉴定。本方法的建立为相关临床病例快速诊断及流行病学调查提供了有效的技术支持。     

Establishment and Application of Multiplex PCR Detection Method for Three Main Gram-negative Bacilli from Livestock and Poultry

To establish a rapid and accurate multiplex PCR method for the clinical detection of three main Gram-negative bacilli from livestock and poultry in Guizhou province,three pairs of specific primers were designed and synthetized according to 23S rRNA gene of Escherichia coli(E.coli),KMT gene of Pasteurella and invA gene of Salmonella.In this study,A mutiplex PCR reaction system was established for detecting the three pathogens at the same time,and the reaction system was optimized and it's performance was evaluated.The results showed that the best concentration of primers was 1.0,1.5and 1.0 μmol/L,respectively,and the best annealing temperature was 56 ℃.The results of performance evaluation showed that the method had good specificity and sensitivity,the sensitivity of Salmonella could reach to 1.44 pg/μL,was the highest.70 Gram-negative clinical isolated strains were detected by the multiplex PCR and it was proofed that the method could identify the three pathogens rapidly and accurately,and it could provide effective technical for the rapid clinical diagnosis and epidemiological survey.     

Follicular fluid and cumulus cells synergistically improve mouse early embryo development in vitro

The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used independently, to improve preimplantation embryo quality in culture. This study was undertaken to evaluate the influence of a cumulus cell monolayer in human follicular fluid on mouse early embryo development in vitro. One-cell embryos were obtained from NMRI mice after superovulation with eCG and hCG. Cumulus cells were prepared from mouse egg-cumulus mass. These cells were separated from red blood cells using a Percoll gradient. Follicular fluid was collected from patients undergoing an IVF program during oocyte pick-up. The cumulus cell monolayer was prepared in follicular fluid (FC) and Ham's F10 (HC). Mouse one-cell embryos were cultured in FC, HC, Ham's F10 (HF) and follicular fluid (FF) for 120 h. Only 10.5% of embryos passed the two-cell block in HF. However, the proportions of embryos passing the two-cell block were 23.1%, 21.4% and 68.5% in FF, HC, and FC treatments, respectively; which were significantly different from HF (p<0.05). The differences between FC and the two other treatments were also significant (p<0.001). In FC, 33.7% of one-cell embryos continued to grow to the blastocyst stage whereas only 2.1% and 1.9% of one-cell embryos in FF and HC reached this stage and no embryos developed to blastocyst in HF. The proportion of blastocysts in FC was significantly higher than all other treatments (p<0.001). It can be concluded that follicular fluid and cumulus cells in monolayer form synergistically improve the early embryo culture condition.     

Development and Application of Multiplex PCR Method for the Drug Resistance Genes Detection in Avian Pathogenic Escherichia coli

To detect the drug resistance genes, the multiplex PCR method for drug resistance genes of β-lactams, tetracyclines, aminoglycosides, amphenicols, and sulfonamides were developed. Based on the sequences of drug resistance genes from GenBank, 17 pairs of specific primers were designed. Then, 4 multiple PCR assays were established through the optimization of PCR reaction conditions and primers concentrations (cat+floR+tetB+tetC; dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1; aac3+aph3+aadA1+strB; tetA+cmlA+strA+sul3). The sensitivity and specificity of these assays were determined. The multiplex PCR assays were used to detect the drug resistance genes of 42 avian pathogenic Escherichia coli (APEC). The drug sensitivity of these APEC strains were also determined, which were compared to the distributions of drug resistance genes in these strains. The results showed that the 17 drug resistance genes were effectively and specifically amplified in these 4 optimized multiplex PCR assays. The detection limits of the 4 multiplex PCR were 10³, 10⁴, 10⁴ and 10⁵CFU of bacteria, respectively. The established multiplex PCR assays are specific and rapid for the detection of drug resistance genes in APEC strains, which showed 92.86% coincident with the drug resistance for these strains. The developed 4 multiplex PCR are simple and rapid assays for drug resistance genes detection, which can be used for the epidemiologic study for drug resistance genes.     

常见耐药基因多重PCR方法的建立及用于禽致病性大肠杆菌耐药性监测

本研究旨在建立β-内酰胺类、四环素类、氨基苷类、酰胺醇类、磺胺类抗菌药物耐药基因的多重PCR检测方法,用于耐药基因的快速检测。根据GenBank公布的上述5类抗菌药物的耐药基因序列,设计17对特异性引物。通过优化PCR体系和反应程序,建立4组耐药基因（cat+floR+tetB+tetC;dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1;aac3+aph3+aadA1+strB;tetA+cmlA+strA+sul3）的多重PCR反应体系。然后,检测多重PCR方法的特异性和敏感性。利用建立的多重PCR方法检测42株禽致病性大肠杆菌的耐药基因,同时,检测其耐药性,比较分析耐药基因和耐药表型之间的相关性。结果显示,建立的4组多重PCR体系可有效扩增出17个耐药基因片段,测序结果表明特异性较好。敏感性结果表明,4组多重PCR体系的菌液敏感性分别为10³、10⁴、10⁴和10⁵CFU。禽致病性大肠杆菌分离株的耐药基因多重PCR和单重PCR检测结果一致,其携带的耐药基因和耐药表型的符合率为92.86%。本研究建立的耐药基因多重PCR方法能简便、快速地检测常见的耐药基因,可用于耐药基因的传播、流行调查。     

牛早期胚胎性别快速鉴别的研究

根据聚合酶链式反应中聚合酶的扩增特性,对PCR反应的循环参数进行研究,取消了PCR反应中延伸这1温度梯度,对2温度梯度PCR方法进行了单重PCR和多重PCR扩增研究,并采用2温度梯度多重PCR方法分别对牛基因组、成纤维细胞、胚胎样品进行了性别鉴别研究,建立了稳定、简便、快速的用于牛早期胚胎性别鉴别的2温度梯度PCR方法.同时还对2温度梯度PCR的扩增能力进行了研究,结果表明：2温度梯度PCR方法能够扩增片段的可能长度为633 bp.     

牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立

为建立检测牛副流感病毒3型（bovine parainfluenza virus type 3,BPIV3）3种基因型的多重RT-PCR方法,根据GenBank上发表的BPIV3 3种基因型病毒株的HN基因序列设计特异性引物,优化反应体系建立多重RT-PCR方法。结果显示,方法可同时扩增出BPIV3 A型150 bp、B型253 bp和C型342 bp的特异性片段,与牛传染性鼻气管炎病毒（IBRV）、牛呼吸道合胞体病毒（BRSV）、牛病毒性腹泻病毒（BVDV）、小反刍兽疫病毒（PPRV）、牛支原体、牛布鲁氏菌、羊布鲁氏菌、牛源多杀性巴氏杆菌A型和B型均无交叉反应,A、B、C基因型BPIV3最低阳性质粒检测量分别为0.89×10⁴、0.92×10⁴和1.53×10⁴拷贝/μL。本试验建立的多重RT-PCR检测方法操作方便、特异性强,应用于临床样本的检测,可快速检测BPIV3 3种基因型。     

哺乳动物胚胎性别鉴定试剂盒的研究

依据牛、山羊、兔、猪等哺乳动物SRY基因高度同源性设计一对22 bp的SRY引物,按照3×2×3因子组合,建立了PCR扩增胚胎DNA最适条件。采用此最适条件扩增了15个羊-兔异种克隆胚胎DNA,结果表明PCR法可以用来鉴别哺乳动物胚胎的性别。采用设计的性别鉴定引物,按照最优PCR扩增胚胎DNA条件配制了PCR性别鉴定试剂盒。     

动物产品及饲料中牛源和羊源性成分三重荧光PCR检测方法的建立

为鉴定和区分饲料及动物产品中牛、山羊、绵羊源性成分,根据线粒体DNA(mitochondrial DNA,mtDNA)种间保守序列,设计合成了3对特异性引物与TaqMan探针,通过对荧光PCR反应体系和反应条件的优化筛选,建立了三重荧光PCR方法,在同一个荧光PCR反应中完成3种动物源性成分的检测。用该方法对16种不同源性的动物DNA进行检测,结果表明能特异地鉴别检测出牛、山羊和绵羊源性成分,且敏感性比现行国标PCR法高100倍。该方法适用于饲料、肉制品、奶制品等动物源性产品的检测。     

动物产品中牛、羊源性成分多重PCR检测方法的建立

以肉骨粉、鱼粉、猪肉干和鱼肉干为研究对象,异硫氰酸胍法提取总DNA,18S rDNA片段的扩增结果表明提取到的DNA中不存在抑制PCR的物质。应用梯度PCR技术对牛、羊源性成分检测的退火温度进行了优化,在单一PCR检测技术的基础上分别进行了18S rDNA片段和牛、羊源性成分的多重PCR分析,得到了预期的结果。试验表明,本文建立的多重PCR方法具有快速、简便、准确等特点,对动物产品牛、羊源性成分检测具有重要意义。