猪繁殖与呼吸综合征RT-LAMP检测方法的建立和初步应用

根据Genbank上已报道的猪繁殖与呼吸综合征病毒(PRRSV)基因组序列设计了2对LAMP引物(P-F3/P-B3、P-FIP/P-BIP),通过反转录-环介导等温扩增技术(RT-LAMP)反应条件的优化,建立了猪繁殖与呼吸综合征病毒(PRRSV)RT-LAMP检测方法,并通过酶切的方法对阳性结果进行了验证。本方法最低可以检测到反应体系内1pg的PRRSV的RNA,表明本方法具有较高的检测灵敏度;通过在反应中加入SYBR-Green I荧光染料后可以方便地用肉眼对试验结果进行可视化判定。所以,本研究建立的PRRSV的RT-LAMP检测方法能够满足基层临床PRRSV病原的快速检测要求。     

可视化的RT-LAMP方法检测禽白血病病毒

本研究旨在建立一种快速检测禽白血病病毒(ALV)的RT-LAMP方法.根据GenBank中ALV序列,在其保守序列区域设计了多套LAMP引物,通过LAMP Real-time Turbidimeter仪对ALV的LAMP引物、反应体系及反应进程进行监测和分析,以评价该方法的特异性和敏感性,并对加入SYBR Green Ⅰ肉眼判定与仪器监测结果进行比对.结果表明:建立的RT-LAMP方法在63℃水浴中36 min可对ALV核酸进行高效扩增,在反应结束后加入SYBR Green I肉眼判断结果与Real-time Turbidimeter仪检测结果一致;该方法具有很强特异性,对其它相关鸡病病原检测结果均为阴性;其检出限量为14.2 pg·μL-1,显示出很高的敏感性;用建立的LAMP方法对24个样品进行检测,结果表明LAMP法与其原检测结果基本一致;本研究建立了可对ALV进行快速检测并可肉眼判定结果的可视化RT-LAMP方法,其操作简便、无需昂贵设备,适用于ALV的快速检测.     

牛轮状病毒RT-LAMP快速检测方法的建立

本研究旨在建立一种快速可视化检测牛轮状病毒(BRV)的反转录-环介导恒温扩增方法(RT-LAMP)。根据BRV的群特异性基因VP6设计一套LAMP引物,对反应条件和试剂浓度进行优化,建立了恒温(63.5℃)、快速(45 min)的检测方法。结果显示:所建立方法特异性好,检测其他对照病毒均为阴性;灵敏度高,最低可检测到1个拷贝的阳性质粒,扩增产物经酶切分析结果正确,扩增结果可通过观察浑浊度或加入染料后直接判定。建立了用于检测BRV的RT-LAMP方法,该方法简便、快速、特异性好、灵敏度高,适合基层和现场检测。     

猪繁殖与呼吸综合征RT-LAMP检测方法研究

应用反转录环介导等温扩增技术(RT-LAMP),利用猪繁殖与呼吸综合征病毒(PRRSV)核衣壳蛋白(N蛋白)ORF7基因保守序列的6个区域设计2对特异引物,经反应体系、反应条件优化及敏感性和特异性试验,建立了PRRSV RT-LAMP检测方法。研究结果表明,该检测方法不需要复杂仪器,仅用一台普通恒温水浴锅,在等温条件(63 ℃)保温1 h,即可完成核酸扩增反应,加入核酸染料SYBR GreenⅠ后,用肉眼即可对试验结果进行准确判定,为田间和基层部门检测猪繁殖与呼吸综合征病毒提供了一种廉价、简便、快速、敏感、特异的新方法。     

实时荧光LAMP法检测转基因猪的标记基因hCD3

利用ESE-Quant tube scanner监测平台,建立了一种基于实时荧光LAMP技术的hCD39基因快速检测方法。针对hCD39基因设计6条特异性引物,在63℃条件下,进行核酸扩增反应45 min。在扩增前加入SYBR GreenⅠ染料作为反应的指示剂,以SYBR GreenⅠ染料的荧光强度作为结果判定标准。该方法灵敏度高,最小可检测到3fg·μL-1;特异性好,与转基因猪中的标记基因hCD46、LEA29Y均无交叉反应;并且可对口腔拭子标本进行检测。因此,研究建立的实时荧光LAMP法,操作简单,反应快速,灵敏度高,特异性好,并且可以对猪活体进行无损伤采样检测,适合在现场快速简便的操作。     

H3N2亚型犬流感病毒RT-LAMP快速检测方法的建立及应用

为了快速诊断H3N2亚型犬流感病毒(CIV)感染,根据H3N2亚型CIV的M和HA基因序列,设计了3组LAMP引物,优化反应条件,建立了H3N2亚型CIV的RT-LAMP检测方法。结果表明,RT-LAMP方法能在63℃恒温条件下,在45min内完成扩增,通过反应管中预加的荧光染料颜色的变化实现目视化判定。该方法对犬的常见病原无交叉反应,具有较好特异性;灵敏度是常规RT-PCR方法的100倍。该方法操作简单、快速灵敏、特异性强,适合现场应用,对H3N2亚型CIV的检测具有实际的应用价值。     

非洲马瘟病毒RT-LAMP检测方法的建立

为了建立非洲马瘟病毒(AHSV)环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,基于AHSV VP7基因保守序列,设计2对特异性扩增引物。通过对反应体系中各组分浓度,反应温度及时间的优化,建立了快速、灵敏的AHSV RT-LAMP检测方法,并对该方法特异性、灵敏度、重复性进行探索。结果表明,在63℃恒温下反应45min,便可进行高效率的特异性扩增。反应产物经琼脂糖凝胶电泳和染料可视化鉴定,能够快速有效检测AHSV。用建立的方法检测马属动物易感的4种疫病病原,结果均为阴性,证实具有较高的特异性。灵敏度是RT-PCR的1 000倍。建立了AHSV RT-LAMP检测方法,具有快速、特异、灵敏、操作简单、设备要求低的特点,适合用于现场AHSV快速检测。     

H3N8亚型马流感病毒RT-LAMP检测方法的建立

为了建立检测H3N8亚型马流感病毒的RT-LAMP方法,根据H3N8亚型马流感病毒HA基因序列,设计了2对LAMP引物,经过优化反应条件,建立了RT-LAMP检测H3N8亚型马流感病毒方法。结果表明,RT-LAMP方法能够在63℃恒温条件下、75min内实现目的基因片段的大量特异性扩增,通过荧光显色就可直接用肉眼判断结果。对H7N7亚型马流感病毒、马动脉炎病毒、马鼻肺炎病毒的核酸无交叉反应,具有较好的特异性;方法的灵敏度比常规RT-PCR的高10倍;该方法操作简便快速、省时省力,而且灵敏度高、特异性强,对H3N8亚型马流感病毒的检测具有实际的应用价值。     

猪繁殖与呼吸综合征病毒LAMP检测方法的建立

应用通用反转录试剂盒和环介导等温核酸扩增技术(LAMP)建立了猪繁殖与呼吸综合征病毒(PRRSV)的快速检测方法,同时评价了该检测方法的特异性、稳定性和灵敏度。结果表明,根据猪繁殖与呼吸综合征病毒M蛋白基因保守区设计的LAMP引物能够在64.5℃60min内实现对目标核酸片段的大量扩增,检测结果可通过直接观察反应副产物(焦磷酸镁)进行判读,该检测系统具有很高的特异性,与猪瘟病毒、猪乙型脑炎病毒等均无交叉反应;通过PRRSV不同毒株疫苗、模拟样品和临床样品确定,该检测系统具有很好的稳定性;通过质粒确定该检测系统可以检测到10拷贝/μL的病毒核酸模板。结果表明成功建立了猪繁殖与呼吸综合征病毒LAMP快速检测方法,为临床提供了一种很好的检测手段。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

中华人民共和国农业部公告 第267号

为贯彻落实《兽药生产质量管理规范》(简称《兽药GMP》),进一步推动兽药GMP实施进程,我部制定了《兽药生产质量管理规范检查验收办法》,现予公告。本公告自2003年6月1日起施行。附件:兽药生产质量管理规范检查验收办法二○○三年四月十日第一章 总则 第一条 为推动《兽药生产质量管理规范》(以下简称兽药GMP)的实施,规范兽药GMP检查验收工作,制定本办法。 第二条 农业部负责全国兽药GMP管理和检查验收工作;负责制修订兽药GMP检查验收管理规定;负责兽药GMP检查员队伍建设和监督管理工作,负责国际兽药贸易中GMP互认工作。 …     

鸡败血支原体垂直传播模式及其阻断方法

以国际标准强毒Ｒ株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离ＭＧ,并收集感染鸡所产蛋分离ＭＧ。结果表明,人工感染４８小时后上、下呼吸道及肺已被全面感染,９６小时气囊已被感染,１２０小时输卵管已能分离到ＭＧ,卵巢始终分离不到ＭＧ。人工感染鸡自１４４小时便能在其所产蛋中分离出ＭＧ。药物治疗能在７２小时内消除感染,油乳剂苗则需２４天后逐渐降低蛋内ＭＧ分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内ＭＧ,但以研制的种蛋浸泡剂药浴效果为最好。     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.