荧光定量PCR技术及其应用研究进展

荧光定量PCR是近年发展起来的一种新的实时定量检测特定核酸技术,它是核酸探针技术、荧光共振能量传递技术和PCR技术的有机结合,而荧光探针是荧光定量PCR的核心.荧光定量PCR具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应争特点,是对原有PCR技术的革新,扩大了PCR的应用范围.论文综述了FQ-PCR技术的原理、FQ-PCR实时定量检测系统及其应用.     

荧光定量PCR技术及其在动物传染病定量检测中的应用

荧光定量PCR是近年发展起来的一 种新的实时定量检测特定核酸技术,它是核 酸探针技术、荧光共振能量传递技术和PCR 技术的有机结合,而荧光探针是荧光定量 PCR的核心。目前报道的荧光探针主要有 TaqMan、Amplisensor、分子信标、Lightcycler 以及在这4种探针基础上发展起来的其他荧 光探针。各种荧光探针在定量PCR中的作 用是识别和报告特定核酸。这些荧光探针与 相应的靶分子杂交时经历一个自发荧光形成 或消失的构象变化,只有与完全互补的靶核 酸杂交时才会出现这种变化,当靶核酸存在 碱基错配或缺失时,荧光探针就不会与之杂 交,也不会出现荧光的变化。在检测时根据 这种荧光变化来确定检测中特定核酸的存在 和量的多少。荧光探针用于定量PCR不但 提高了PCR的检测灵敏度,还能在同一封闭 管中对模板进行准确定量检测,特别适合特 定核酸扩增的实时监测。荧光定量PCR用 于动物传染病的诊断,不但能定量检测病原 体感染的强弱和在机体内的分布,还具有灵 敏、快速、省力的特点。     

鸭坦布苏病毒TaqMan荧光定量PCR方法的优化

为提高鸭坦布苏病毒(DTMUV)TaqMan荧光定量PCR方法的敏感性和简化反应条件,本研究在前期建立的TaqMan荧光定量PCR方法的基础上,设计一条特异性的反转录引物,优化反应体系,建立了简便、快速、敏感的TaqMan荧光定量PCR方法。该荧光定量PCR方法最低检测限为10拷贝,敏感性是未优化的荧光定量PCR方法的5倍、是普通PCR方法的100倍,并且该方法对DTMUV的最低检测限是0.01半数鸡胚致死量(ELD50)。通过批内和批间实验的变异系数表明优化的荧光定量PCR方法的重复性比未优化的荧光定量PCR方法好。通过该优化的荧光定量PCR方法检测其它常见的鸭病病毒的DNAs或RNAs,证明了该方法特异性好。对现地60份疑似DTMUV的样品进行检测,用该方法检出57份样品为阳性,明显高于普通PCR,并且在区分35Ct值左右样品试验中敏感性优于未优化的荧光定量PCR方法。该优化的DTMUV TaqMan荧光定量PCR方法更适用于DTMUV的快速定量流行病学诊断。     

鹅GHR基因荧光定量PCR险测方法的建立

采用PCR方法克隆了鹅生长激素受体(GHR)基因,构建了重组质粒pMD-18T-GHR,并将其作为标准阳性模板.通过对反应条件进行优化,以定量的10倍系列稀释的质粒pMD-18T-GHR为标准品进行荧光定量PCR扩增,建立了检测GHR的荧光定量PCR方法.结果显示,该方法构建的标准曲线线性关系良好,建立的GHR基因荧光定量PCR检测方法灵敏度高、特异性强(可以检测出低于10个拷贝/μL的样品),准确可靠.籽鹅和莱茵鹅GHRmRNA出壳到90日龄生长过程中肝脏中的表达量不同,30日龄不同组织表达量各有变化特点.     

鹅GHR基因荧光定量PC凡检测方法的建立

采用PCR方法克隆了鹅生长激素受体（GHR）基因,构建了重组质粒pMD-18T-GHR,并将其作为标准阳性模板。通过对反应条件进行优化,以定量的10倍系列稀释的质粒pMD-18T-GHR为标准品进行荧光定量PCR扩增,建立了检测GHR的荧光定量PCR方法。结果显示,该方法构建的标准曲线线性关系良好,建立的GHR基因荧光定量PCR检测方法灵敏度高、特异性强（可以检测出低于10个拷贝／μL的样品）,准确可靠。籽鹅和莱茵鹅GHRmRNA出壳到90日龄生长过程中肝脏中的表达量不同,30日龄不同组织表达量各有变化特点。     

荧光定量PCR技术在分子检测上的研究进展

荧光定量PCR技术是近年来发展起来的一种核酸定量技术,因其特异性强、灵敏度高、重复性好、定量准确等优点成为核酸检测中的重要工具,作者将其研究进展作一综述,以供参考。     

基于Taq Man探针的猪繁殖与呼吸综合征病毒荧光定量PCR检测方法的研究

通过条件优化,以定量的10倍系列稀释的质粒pMD-ORF7为标准品进行荧光定量PCR扩增并制作标准曲线建立了PRRSV的荧光定量RT-PCR检测方法.结果表明,该方法检测灵敏度可达1.0×100拷贝/μL;用该方法对6份不同的组织样品进行重复性检测.结果显示具有良好的重复性和重现性.对阳性组织病料的检测表明该方法与常规RT-PCR阳性符合率为100%.但检测灵敏度高出常规RT-PCR 100倍.     

应用TaqMan荧光定量PCR检测H3N8亚型马流感病毒

为建立马流感病毒(ETV)的检测方法,本试验针对H3N8亚型ETV血凝素(HA)基因高度保守序列设计并合成了2对引物和1条TaqMan荧光探针,建立了TaqMan荧光定量PCR方法.经用TaqMan荧光定量PCR、RT-PCR和病毒分离方法分别检测新疆等省135份疑似EIV马鼻拭子样品,其结果表明:3种方法的马流感检出率分别为54.07%、37.78%、0.89%;TaqMan荧光定量PCR可检出马流感病毒基因组RNA的灵敏度可达10拷贝/反应.而且与其他马呼吸道病毒均无交叉反应,具有良好的特异性、敏感性和重复性.该方法为H3N8亚型马流感的早期诊断及分子流行病学调查等提供了一种新的快速、准确的定量检测技术.     

兔病毒性出血症快速检测方法的建立

兔病毒性出血症是兔的1种传播性极强、致死率极高的重要传染病,针对性地加强该病原的进出境监测将对动物疫病防控具有重要的意义。本研究使用软件分析设计RT-PCR、实时荧光定量RT-PCR的引物和TaqMan探针,建立了兔病毒性出血症RT-PCR和实时荧光定量RT-PCR方法,并测定其特异性和敏感性。试验结果显示,只有阳性参照样品的DNA出现扩增,其他对照样品均未见扩增产物,证实这些检测方法具有良好的特异性。对阳性样品DNA进行10倍梯度稀释,再分别用新建的2种方法进行试验,结果显示兔病毒性出血症RT-PCR检测的DNA下限量为100 pg,实时荧光定量RT-PCR的DNA下限量为100 fg,2种方法都具有较高的敏感性。新建的RT-PCR和实时荧光定量RT-PCR方法互补应用,有助于缩短检疫周期、提高检测效率,为出入境口岸部门加强入境试验兔或其他不同用途兔的疫病检测提供技术保障。     

基于M基因的新城疫病毒实时荧光定量RT-PCR的建立及其对临床样品中新城疫病毒检测的研究

根据新城疫病毒M基因的保守序列,设计合成1对引物和TaqMan探针,以作者实验室构建并保存的鹅源新城疫病毒zJ1株M基因阳性重组质粒为标准品模板建立荧光定量PCR标准曲线,结合ABI公司的7300型荧光定量PCR仪,建立了一种敏感、特异、重复性好的快速检测新城疫病毒核酸载量的TaqMan荧光定量RT-PCR方法.该方法在106～101拷贝范围内具有良好的线性关系,可检测到初始模板中3拷贝·μL-1的病毒核酸,与传统的病毒分离方法具有相近的敏感性,二者对500份临床禽泄殖腔棉拭样品检测结果阳性数、阴性数符合率分别为90.0%、99.8%.经临床应用表明:荧光定量PCR的建立为早期诊断禽新城疫病毒、定量分析禽新城疫病毒感染程度奠定了基础.     

Viability of Fresh and Frozen-Thawed Biopsied Bovine Embryos

Gustafsson H., U. Jaakma and M. Shamsuddin: Viability of fresh and frozen-thawed biopsied bovine embryos. Acta vet. scand. 1994,35,217-222.– Bovine embryos were biopsied using a simplified splitting technique and frozen-thawed according to a standard method with glycerol as cryoprotectant. The viability of fresh and frozen-thawed biopsied and intact embryos were evaluated after in vitro culture, by means of fluorescence test or following transfer to recipients. The survival rates after in vitro culture of fresh intact and biopsied embryos and of frozen-thawed intact and zona free embryos were not significantly different (70%, 60%, 68% and 52%, respectively), but significantly reduced for bipsied frozen-thawed embryos (16%) (p≤0.05). The pregnancy results after transfer of biopsied frozen-thawed embryos were also significantly lower (8%) compared to fresh biopsied embryos (39%) (p≤0.05). Both intact and biopsied embryos fluoresced after incubation with diacetylfluorescin but with higher intensity for the intact embryos. It is suggested that the reduced survivability for the frozen-thawed biopsied embryos might be caused by combined effects of the loss of the zona pellucida and the reduction of cells as a result of the simplified biopsy technique. It is concluded that improved biopsy and/or freezing techniques must be used if biopsied embryos have to be frozen.     

关于推进中国奶牛全混合日粮饲养技术的思考

全混合日粮（total mixed ration ,TMR）饲养技术的使用能够保证奶牛摄入均衡的营养,节省大量的人力和物力,并提高奶牛的生产性能,在中国加快推进TMR饲养技术有着重要的现实意义。而中国现阶段奶牛的饲养模式主要以小规模养殖为主,由于缺乏资金和技术支持,大多数中、小型奶牛场、养殖户还不能使用TMR饲喂技术来提高奶牛生产性能。TMR配送服务是由专门的配送公司集中生产不同饲养阶段的TMR日粮,再提供给周边养牛户的一种生产配送形式。此种方式可以使小规模奶牛养殖户也能用到TMR饲喂技术,从而提高奶牛生产水平,增加养殖者收益,并且推进TMR技术在中国的推广和应用。     

Pocket technique or pocket technique combined with modified orbital rim anchorage for the replacement of a prolapsed gland of the third eyelid in dogs: 353 dogs

The aim of this retrospective study was to evaluate the results obtained in 353 dogs (420 eyes) using two different surgical techniques for correction of a prolapsed gland of the third eyelid: the Morgan's pocket technique and a technique combining Morgan's approach with a slightly modified periosteal anchoring technique of Stanley and Kaswan. The pocket technique was used in 234 eyes and the combined technique in 186 eyes. Successful repositioning was obtained in 95% of all cases, with recurrence occurring in 5%. The recurrence rate in large breed dogs such as the English Bulldog and Boxer was lower with the combined technique than with the pocket technique.     

RNA病毒感染性分子克隆的研究

随着反向遗传学技术的兴起和发展,RNA分子体外操作已经成为可能,并且技术日臻完善。 反向遗传操作技术的关键就是构建RNA病毒的感染性分子克隆,在DNA分子水平上对其进行体外操作,从而研究病毒结构与功能的方法。作者对感染性克隆的构建方法及感染性体外转录方法作一综述。      

Veterinary decision making in relation to metritis - a qualitative approach to understand the background for variation and bias in veterinary medical records

Background

Results of analyses based on veterinary records of animal disease may be prone to variation and bias, because data collection for these registers relies on different observers in different settings as well as different treatment criteria. Understanding the human influence on data collection and the decisions related to this process may help veterinary and agricultural scientists motivate observers (veterinarians and farmers) to work more systematically, which may improve data quality. This study investigates qualitative relations between two types of records: 1) ''diagnostic data'' as recordings of metritis scores and 2) ''intervention data'' as recordings of medical treatment for metritis and the potential influence on quality of the data.

Methods

The study is based on observations in veterinary dairy practice combined with semi-structured research interviews of veterinarians working within a herd health concept where metritis diagnosis was described in detail. The observations and interviews were analysed by qualitative research methods to describe differences in the veterinarians'' perceptions of metritis diagnosis (scores) and their own decisions related to diagnosis, treatment, and recording.

Results

The analysis demonstrates how data quality can be affected during the diagnostic procedures, as interaction occurs between diagnostics and decisions about medical treatments. Important findings were when scores lacked consistency within and between observers (variation) and when scores were adjusted to the treatment decision already made by the veterinarian (bias). The study further demonstrates that veterinarians made their decisions at 3 different levels of focus (cow, farm, population). Data quality was influenced by the veterinarians'' perceptions of collection procedures, decision making and their different motivations to collect data systematically.

Conclusion

Both variation and bias were introduced into the data because of veterinarians'' different perceptions of and motivations for decision making. Acknowledgement of these findings by researchers, educational institutions and veterinarians in practice may stimulate an effort to improve the quality of field data, as well as raise awareness about the importance of including knowledge about human perceptions when interpreting studies based on field data. Both recognitions may increase the usefulness of both within-herd and between-herd epidemiological analyses.     

A literature review of reports of the stability and storage of common injectable chemotherapy agents used in veterinary patients

Many chemotherapy drugs used in human patients are discarded after single use or within 24 h of reconstitution, as per the manufacturer's product label recommendations. This can be wasteful and costly to veterinary clients. This report reviews the published stability and storage data for 19 injectable chemotherapy drugs commonly used in veterinary medicine. Based on these data, storage procedures are presented, assuming aseptic technique and a closed system drug transfer device (CSDTD) are used for drug preparation and handling. Further studies on the risk of microbiological contamination of chemotherapeutics using a CSDTD, and validated high quality drug assays such as stability‐indicating high‐performance liquid chromatography, are required. The authors' intent is not to supersede product label recommendations, but to suggest that longer storage without significant loss of drug efficacy may be possible, thus reducing the costs of chemotherapeutics to some veterinary clients.     

石墨烯膜不同加温方式对设施高架基质草莓生长结果的影响

为探究石墨烯电热膜不同加温方式对草莓生长和结果的影响,本文以红颜草莓为试材,采用草莓顶部、基质表面和根系周边组合的7种加温方式,以电热线加温为对照,研究了不同石墨烯加温方式对设施高架基质草莓植株、叶片、果实和根系等相关性状的影响。结果表明：石墨烯膜放置草莓根系附近;石墨烯膜放置于根系附近和顶部20cm处组合;石墨烯膜放置于根系附近、基质表面和植株顶部20cm处组合在各个性状表现均优于其他处理,但石墨烯膜放置于根系附近和顶部20cm处组合;石墨烯膜放置于根系附近、基质表面和植株顶部20cm处组合耗电量相对较高,石墨烯膜放置草莓根系附近处理耗电量相对较低,可以作为石墨烯的加温方式进行推广应用。     

不同咸蛋腌制温度对禽流感毒灭活的影响

经人工接种H9亚型禽流感病毒(AIV)的鸭蛋,分别在12℃、22℃和32℃条件下按常规方法腌制于饱和盐水中,同时以置饱和盐水中的禽流感病毒和未经处理禽流感病毒样品作为对照,通过MDCK细胞培养、间接免疫荧光方法定期进行禽流感病毒毒力测定,结果在12℃、22℃和32℃条件下腌制鸭蛋中的禽流感病毒分别于49天、27天和4天失去毒力,置饱和盐水中的禽流感病毒分别在27天、9天和2天失去毒力,而未经处理的禽流感病毒分别在92天、29天和17天失去毒力;不同试验温度条件下,以荧光RT-PCR检测腌制鸭蛋和对照样品中的禽流感病毒,在100天后仍可检出病毒核酸(Ct<30)。以上检测结果表明,在腌制鸭蛋时于常温下置饱和盐水腌制40天以上的传统咸蛋生产工艺,可使禽流感病毒完全失去毒力,腌制的鲜咸蛋携带或传播禽流感病毒的风险极低或不存在风险。     

葡萄风信子花青素3-O-葡糖基转移酶基因的克隆及其表达分析

花青素3-O-葡糖基转移酶(3GT)是花青素苷合成最后一步的关键酶,可把不稳定的花青素催化成花青素苷。本研究利用PCR技术从蓝色葡萄风信子‘亚美尼亚’(Muscari armeniacum)中克隆到一个3GT基因(Ma3GT),Ma3GT cDNA全长1 377bp,编码458个氨基酸,与其他植物的3GT蛋白序列相似性达55%~64%。进化分析表明,Ma3GT与单子叶植物3GT聚为一类,与小苍兰(Freesia hybrida)、荷兰鸢尾(Iris hollandica)3GT亲缘关系最近。荧光定量PCR分析发现,Ma3GT在‘亚美尼亚’花(S_4)中高表达,在叶片中微量表达,根和鳞茎中几乎不表达,在‘白丽人’及‘粉日出’各个组织中均不表达。在‘亚美尼亚’中,Ma3GT的转录表达受花发育调控,开花早期(S_1)几乎不表达,随着花发育表达量逐渐增加,至完全开放的时期(S_4)达到最高峰。此外,Ma3GT在不同品种的花中转录表达不同,在蓝色品种‘亚美尼亚’中高表达,而在白色品种‘白丽人’和粉色品种‘粉日出’中几乎不表达。本研究为进一步研究Ma3GT基因在葡萄风信子花瓣呈色中的功能及其花色分子育种提供基因资源和依据。     

南瓜状晚棱脐橙芽变的研究

现获得晚熟脐橙品种‘晚棱脐橙’一芽变材料，果皮从果蒂到果顶方向有突起棱条。流式细胞仪检测发现南瓜状晚棱脐橙芽变与‘晚棱脐橙’同为二倍体。不同时期果实常规品质分析发现南瓜状晚棱脐橙芽变果实略呈扁圆形，维生素C含量较高，酸甜适中，成熟期晚于‘晚棱脐橙’。石蜡切片分析发现南瓜状晚棱脐橙芽变与‘晚棱脐橙’果皮显微结构发现二者存在细胞水平上的差异。应用ISSR分子标记技术对南瓜状晚棱脐橙芽变与‘晚棱脐橙’进行遗传鉴定，通过正交试验设计，建立反应优化体系。从100条ISSR引物中筛选出17条能扩增出清晰度高、多态性好的引物，对南瓜状晚棱脐橙芽变与‘晚棱脐橙’的基因组DNA进行ISSR-PCR扩增，结果表明有9条引物可使南瓜状晚棱脐橙芽变与‘晚棱脐橙’在某些扩增位点上出现多态性，说明南瓜状晚棱脐橙芽变与‘晚棱脐橙’在DNA分子水平上存在明显差异。