基于便携式荧光定量PCR仪的猪流行性腹泻病毒和传染性胃肠炎病毒现场检测方法的建立及应用

为建立猪流行性腹泻病毒和传染性胃肠炎病毒一步法荧光RT-PCR鉴别检测方法。本研究参照Gen Bank中猪流行性腹泻病毒以及传染性胃肠炎病毒特异性基因序列,设计特异引物和Taq Man探针,通过优化反应条件,建立了检测猪流行性腹泻病毒和传染性胃肠炎病毒的一步法荧光RT-PCR方法,并验证该方法的特异性、敏感性、重复性。结果表明,该方法检测猪流行性腹泻病毒和传染性胃肠炎病毒灵敏度分别可达0.32 TCID_(50)/100μL和1.58 TCID_(50)/100μL,该法对猪轮状病毒(RV)、猪瘟病毒(CSFV)、猪繁殖和呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)的检测结果均为阴性。本试验建立的Taq Man一步法荧光定量RT-PCR检测方法可对猪流行性腹泻和传染性胃肠炎进行快速诊断,适合现场检测,为猪病毒性腹泻的诊断和防控奠定了基础。     

欧洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立

根据欧洲型猪繁殖与呼吸综合征病毒(PRRSV)基因序列,设计E1和E6 2套PCR引物和TaqMan荧光探针,建立E1和E6荧光RT-PCR方法,用于检测PRRSV。反应条件优化后,用10倍倍比稀释的质粒DNA、cDNA进行PCR扩增以检测其灵敏度,同时对猪瘟(CSFV)、猪伪狂犬(PRV)等7种病毒进行特异性检测。结果显示,建立的E1和E6荧光RT-PCR可以检出欧洲型PRRSV,敏感性依次为616和216个拷贝的PRRSV重组质粒DNA,而CSFV等非PRRSV均为阴性。建立的E1、E6荧光RT-PCR具有快速、灵敏、准确、低污染等优点,可作为检测欧洲型PRRSV的技术储备。     

食源性动物组织中CSFV和PRRSV双重双色荧光定量RT-PCR检测方法的建立及应用

为建立同时检检测食源性动物组织中猪瘟病毒(CSFV)和猪蓝耳病病毒(PRRSV)的双色荧光定量RT-PCR方法。本研究根据SCFV和PRRSV基因序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应的体系和扩增条件,建立了能够检测食源性动物组织中SCFV和PRRSV的双重双色荧光定量PCR的方法。其检测下限为1×102拷贝/μL,而且与其他一些猪病病毒无交叉反应,具有良好的特异性。该方法重复性和稳定性试验表明其组内和组间的变异系数最高分别为4.2和4.5。对比试验表明,该方法对CSFV和PRRSV检验的敏感性为常规RT-PCR方法的200倍;该方法的建立为食源性动物组织中CSFV和PRRSV提供了有效手段,该方法特异性和敏感性较好,能够应用于临床检测。     

变异型猪繁殖与呼吸综合征病毒TaqMan荧光定量RT-PCR方法的建立与初步应用

参照GenBank的变异型猪繁殖与呼吸综合征病毒(PRRSV)与普通PRRSV的Nsp2段基因序列,设计并合成1对引物和TaqMan探针。通过对反应条件优化,标准质粒构建,建立了TaqMan荧光定量RT-PCR诊断变异型PRRSV方法。结果表明,该方法具有特异性强,敏感性高等特点,能够检测出264个拷贝数的标准质粒品,0.562 3TICD50的病毒量,比RT-PCR敏感10倍。对22份病料样品进行检测,结果有8份为阳性,阳性率为36.4%。由于该方法具有定量、快速、准确、敏感等优点,适用于对猪群感染变异型猪繁殖与呼吸综合征(PRRS)早、中、晚期的诊断,对有效诊断及防治高致病性PRRS的发生起到重要作用。     

双重RT-PGR方法检测猪流感病毒和猪繁殖与呼吸综合征病毒

为建立特异、敏感的猪流感病毒(SIV)和猪繁殖与呼吸综合征病毒(PRRSV)的双重RT-PCR检测方法,本研究根据GenBank登录的SIV M基因保守序列和PRRSV美洲型毒株的N基因保守序列,设计合成了2对特异引物,通过对扩增条件的优化,建立检测SIV和PRRSV的双重RT-PCR方法.检测结果显示:该方法可同时扩增出SIV(345 bp)和PRRSv(520 bp)的特异性片段;而猪瘟病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型及阴性鸡胚尿囊液核酸扩增结果均为阴性;对SIV和PRRSV 2种病毒混合液的最小检出量分别为102 EID50/0.1 mL和103TCID50/0.1 mL.应用双重RT-PCR和病毒分离法对12份临床疑似样品进行对比检测,结果表明:除双重RT-PCR检测到双阳性的3份混合感染病料中1份未分离到PRRSV外,其余2份均分离出病毒.证明该方法具有良好的特异性、敏感性,可以用于临床样品的早期快速检测.     

猪蓝耳病病毒变异株TaqMan探针实时荧光定量RT-PCR鉴别诊断方法的建立

根据GenBank收录的PRRSV基因序列,设计一对引物F、R和一条TaqMan荧光探针P,建立了猪蓝耳病病毒荧光RT-PCR检测方法.该方法敏感性比常规的RT-PCR高100倍,对PRRSV细胞培养物的检测下限可以达到2个TCID50,能特异检测出蓝耳病病毒,并且可以鉴别诊断PRRSV变异株和经典株,同时根据灵敏度试验建立的标准曲线,能够对PRRSV RNA进行相对定量.     

鉴别欧洲型和美洲型PRRSV荧光定量RT-PCR方法的建立

为了建立鉴别欧洲型和美洲型猪繁殖与呼吸综合征病毒(PRRSV)的荧光定量RT-PCR方法,根据欧洲型和美洲型PRRSV的M基因保守序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应体系和扩增条件,建立了能够鉴别欧洲型和美洲型PRRSV的双重双色荧光定量RT-PCR方法。该方法的重复试验表明其组内和组间的变异系数最高分别为1.30%和1.96%;灵敏性及特异性试验表明其检测下限为1×101copies/μL,而且与其他一些猪源病毒无交叉反应,具有良好的特异性。该检测方法的建立为不同型别的PRRSV快速诊断提供了有效手段,能够应用于临床样品检测。     

高致病性猪繁殖与呼吸综合征病毒双重液相基因芯片检测方法的建立

为了实现快速检测猪繁殖与呼吸综合征病毒(PRRSV)并同步鉴别高致病性PRRSV毒株(HPPRRSV),根据PRRSV囊膜蛋白GP2基因保守序列和HP-PRRSV特有的Nsp2基因区保守序列设计特异扩增引物和杂交探针,通过双重一步法RT-PCR不对称扩增和双重微球杂交反应,建立了双重液相基因芯片方法。对12株PRRSV以及其他12种猪病原体的检测显示,该法能特异性检测12株PRRSV毒株并准确鉴别其中7株HP-PRRSV,与其他病原体无交叉反应;对PRRSV、HP-PRRSV病毒液的检测低限均小于每个反应0.5TCID_(50);其组内、组间检测变异系数均10%;检测55份疑似临床样品并与商品化荧光RTPCR试剂盒比较检测结果,符合率达98.2%(54/55)。研究结果为适应临床快速检测PRRSV提供了一种新的分子生物学检测方法。     

猪繁殖与呼吸综合征病毒和猪流行性腹泻病毒双重TaqMan实时荧光定量RT-PCR检测方法的建立

为了建立快速、简便且能同时检测猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)和猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的方法,试验根据GenBank中已登录的PRRSV ORF7基因序列和PEDV N基因序列,设计2对特异性引物和2条TaqMan探针,通过优化扩增条件建立检测PRRSV和PEDV的双重TaqMan实时荧光定量RT-PCR方法,同时还进行了特异性、敏感性、重复性试验以及临床样品检测。结果表明:最终确立的20μL扩增体系中各引物和探针的加样量分别为:PRRSV-F 0.8μL,PRRSV-R 0.6μL,PRRSV-P 0.5μL;PEDV-F 1.2μL,PEDV-R 1.0μL,PEDV-P 0.7μL。(优化后的最佳扩增程序为:50℃反转录6 min;95℃预变性1 min;95℃变性10 s,55℃退火10 s,72℃延伸10 s,共40个循环)。检测PRRSV和PEDV的敏感性可分别达到1.05 TCID_(50)/100μL和3.16 TCID_(50)/100μL,该方法特异性好,不与其他常见猪病病原体发生交叉反应,批内变异系数和批间变异系数均不高于2.0%。用该方法对234份临床样品进行检测,PRRSV阳性样品15份, PEDV阳性样品20份,检出率高于常规RT-PCR方法。说明试验建立的双重TaqMan实时荧光定量RT-PCR方法敏感性高,特异性好,可同时准确、快速检测PRRSV和PEDV。     

基于盖他病毒nsP3基因的TaqMan荧光定量RT-PCR检测方法的建立及初步应用

为建立一种灵敏、特异且高效的检测盖他病毒(GETV)的TaqMan荧光定量RT-PCR方法，本研究根据GETV的nsP3基因设计特异性引物及探针，以GETV (SC483株) cDNA作为模板，扩增目的基因并克隆至p ET-29a载体中，构建重组质粒标准品p ET29a-nsP3并经PCR和测序鉴定。基于该质粒标准品，采用方阵法优化引物、探针浓度以及退火温度，初步建立了一种基于GETV nsP3基因的TaqMan荧光定量RT-PCR检测方法，并绘制标准曲线。以GETV (SC483株、SC266株)、辛德毕斯病毒(SINV)、猪流感病毒(H3N2、SIV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪繁殖和呼吸障碍综合征病毒(PRRSV)等病毒的基因组RNA反转录为cDNA作为模板，利用本实验建立的TaqMan荧光定量RT-PCR方法检测，结果显示，仅GETV为阳性，SINV、SIV、TGEV、PEDV、PRRSV均为阴性，表明该方法特异性较强；分别以10倍倍比稀释(3.07×10^-1拷贝/μL～3.07×10⁸拷贝/μL...     

H9N2亚型禽流感病毒传播途径特性的比较及其表面基因序列分析

选择中国大陆最早分离的H9N2亚型禽流感病毒(avian influenza virus,AIV)A/Chicken/Guangdong/SS/94(H9N2)(缩写为SS株)和1998年大流行时期分离的H9N2亚型AIVA/Chicken/Shanghai/F/98(H9N2)(缩写为F株)为研究对象,对其在SPF鸡体内的复制能力和传播途径特性比较后发现,F株在4周龄SPF鸡气管中的复制能力高于SS株,F株可以经气溶胶传播途径传播,SS株不能经气溶胶传播途径传播;利用反转录-聚合酶链反应(RT-PCR)方法获取F株和SS株的HA和NA基因的cDNA,序列分析得知,F株和SS株的HA和NA基因的同源性分别是96.6%和98.1%;HA基因的裂解位点氨基酸序列都是PARSSR↓GL,但有5个氨基酸的差异,即166位N(F)→D(SS)、198位A(F)→V(SS)、217位V(F)→I(SS)、335位G(F)→R(SS)、504位L(F)→S(SS);2株病毒的NA基因在63~65位都存在氨基酸缺失,但在NA基因红细胞吸附位点的氨基酸序列不同,分别是IKKDSRSG(F)和IKEDLRSG(SS)。F株和SS株的传播特性差异是否与其表面基因序列有关,有待进一步研究。     

禽类的起源、演化及我国主要家禽品种类型与分布

家禽是重要经济价值动物.本文从禽类种群进化学说出发,简介了禽类的起源、演化、动物学分类和家禽的驯化(养)与品种的形成,并对我国主要家禽(鸡、鸭、鹅)地方品种和培育品种(配套系)的分布与类型作了描述,以期为研究我国家禽起源系统,保护与利用我国家禽品种,促进家禽生产可持续发展提供参考.     

一起猪链球菌病和伪狂犬病的混合感染的诊治

近年以来,由于市场因素的刺激,生猪的存养量大幅上升,再加上由于流通环节较多,流通非常频繁,流通距离越来越远。这对繁荣经济,增加养殖效益起了重要的推动作用,但也同时给疾病的感染和传播创造了有利条件,给猪病的防治带来了困难。有的猪场感染了传染病后,由于治疗不及时不得法,而造成了惨重的经济损失。2008年7月中旬,我街道一养猪户因盲目从外地购进中猪,发生猪病疫情,引起猪只连续死亡,造成一定的经济损失。根据流行病学、临床症状、剖检变化和实验室诊断,诊断该病为猪链球菌病和猪伪狂犬病混合感染,现报告如下。     

秋冬季预防和治疗虫媒性传染病鸡白冠病和鸡痘

１前言１．１鸡白冠病鸡白冠病是由卡氏住白细胞原虫寄生于鸡的红细胞和单核细胞而引起的鸡的贫血性疾病。吸血昆虫蚋和库蠓叮咬鸡引起传播，是主要的传播媒介，一般在夏末和秋季多发，由于夏季降雨量较大，部分沟渠积水，库蠓和蚋多孳生，因此在多雨水涝的年份发病率明显增高。１９９８年中国从南到北发生洪涝灾害，吸血昆虫的孳生格外严重，出现了一个白冠病多发年，而后两年发病稍轻，并有地区性，今年８月中旬以来白冠病的发病呈抬头趋势，有一定的死亡率，对蛋鸡产蛋率也会引起一定程度的降低，应引起养鸡户的重视。１．２鸡痘鸡痘也是…     

Estimations of repeatability and heritability of egg albumen antimicrobial activity and of lysozyme and ovotransferrin concentrations

1. The repeatability and heritability of growth inhibition by egg albumen of two major pathogenic bacteria, a Gram-negative (Salmonella Enteritidis) and a Gram-positive (Staphyloccocus aureus) and of two antimicrobial albumen proteins, lysozyme and ovotransferrin, were estimated in commercial pedigree hens. 2. Repeatability was evaluated in 100 egg-type hens at the beginning, middle and end of the laying cycle on eggs collected for 3 weeks. Heritabilities were estimated at 36 to 40 weeks of age on 400 pedigree hens (2 eggs/hen), which were the offspring of 25 sires each mated with 4 dams. Ovotransferrin and lysozyme were quantified by ELISA. Salmonella Enteritidis (S.E.) and Staphyloccocus aureus (S.A.) were inoculated into a sample of sterilised albumen and enumerated after incubation. 3. Total protein content in albumen decreased with age of laying hens, whereas there were increases in lysozyme or ovotransferrin concentrations and in the bacteriostatic effect of albumen. 4. Repeatability for bacterial growth in albumen ranged from 0.29 to 0.39 for the number of S.E. (log cfu/ml) one day post inoculation (p.i.) but was lower and more variable at 5 d p.i. or for S.A. number. It ranged from 0.27 to 0.38 for S.E. and S.A. number at the mid period of the laying cycle. Repeatabilities were low and variable for total egg albumen protein or lysozyme and ovotranferrin concentrations (0 to 0.22). 5. Negative phenotypic correlations were observed between lysozyme concentrations and S.E. number but that between lysozyme and S.A. number was not significant. 6. Heritabilities were low (0.01 to 0.09) for protein traits. They were 0.11 for S.A. number and 0.16 for S.E. number one day p.i. 7. It appears to be more efficient to select on global bacterial growth than on specific antimicrobial proteins. The most promising trait is the number of S.E. one day p.i.     

种鹅矛形剑带绦虫与背孔吸虫混合感染的诊治

2005年9月份,大庆市红岗区个体养鹅专业户送检6只病死的5月龄左右隆昌鹅和长白鹅,经过实验室诊断确诊为矛形剑带绦虫与背孔吸虫混合感染。矛形剑带绦虫属膜壳科     

Evaluation of lysozyme and lactoferrin in lacrimal and other ocular glands of bison and cattle and in tears of bison

OBJECTIVE: To evaluate lactoferrin and lysozyme content in various ocular glands of bison and cattle and in tears of bison. SAMPLE POPULATION: Tissues of ocular glands obtained from 15 bison and 15 cattle and tears collected from 38 bison. PROCEDURE: Immunohistochemical analysis was used to detect lysozyme and lactoferrin in formalin-fixed, paraffin-embedded sections of the ocular glands. Protein gel electrophoresis was used to analyze ocular glands and pooled bison tears by use of a tris-glycine gel and SDS-PAGE. Western blotting was used to detect lactoferrin and lysozyme. RESULTS: Immunohistochemical staining for lactoferrin was evident in the lacrimal gland and gland of the third eyelid in cattle and bison and the deep gland of the third eyelid (Harder's gland) in cattle. Equivocal staining for lactoferrin was seen for the Harder's gland in bison. An 80-kd band (lactoferrin) was detected via electrophoresis and western blots in the lacrimal gland and gland of the third eyelid in cattle and bison, Harder's glands of cattle, and bison tears. An inconsistent band was seen in Harder's glands of bison. Lysozyme was not detected in the lacrimal gland of cattle or bison with the use of immunohistochemical analysis or western blots. Western blots of bison tears did not reveal lysozyme. CONCLUSIONS AND CLINICAL RELEVANCE: Distribution of lactoferrin and a lack of lysozyme are similar in the lacrimal gland of cattle and bison. Differences in other tear components may be responsible for variability in the susceptibility to infectious corneal diseases that exists between bison and cattle.     

Description and classification of abnormal cercariae of Schistosoma mattheei and a method of concentration and collection

Over a period of about 12 years, 30 abnormal Schistosoma mattheei cercariae were found among a total of approximately 2.8 million examined. Initially seven were recovered from about 1.02 million (0.0007%), which were examined individually while being counted with the aid of a stereoscopic microscope. Subsequently, on the strength of relatively high percentages of abnormal individuals recovered when counting cercariae that failed to penetrate into oxen, it appeared that the morphologically abnormal cercariae were unable to swim and would mostly sediment out of a suspension while most of the normal cercariae would remain swimming. This surmise is supported by recovery of 23 morphologically abnormal cercariae (0.001%) from about 1.8 million, by examining the sediment after the cercarial suspension had been left standing undisturbed in glass measuring cylinders. The abnormalities ranged from aberrant tails only (e.g. an underdeveloped tail, or different degrees of schism) or aberrant heads only, to abnormalities of both the heads and tails. A suggested schematic classification of abnormal cercariae is presented. A young, adult hamster was exposed to eight S. mattheei cercariae with complete schism of the shaft of the tail, by pipetting the cercariae onto the shaved abdominal skin of the anaesthetised animal. Two underdeveloped females were subsequently encountered in squash preparations of the liver when the hamster was killed for worm recovery 10 weeks after infection, thus showing that some of the abnormal cercariae were viable. A method is also described for killing and fixing cercariae while retaining some of the shining brilliance of live cercariae, without them becoming shrivelled, granular and semi-opaque, as occurs when cercariae die spontaneously or are killed with heat. This is apparently the first report of abnormal cercariae of S. mattheei. In addition, a method of concentrating abnormal cercariae after emergence from a snail, a schematic classification of abnormal cercariae and a method for killing and fixing cercariae while retaining much of the shiny brilliance of live cercariae are also reported for the first time as far as is known.