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1.
为了研究猪乙型脑炎病毒(Japanese encephalitis virus, JEV)在仓鼠肾细胞(BHK-21细胞)中的增殖情况，试验首先建立了实时荧光定量PCR方法，并检测该方法的敏感性、特异性和重复性，然后利用建立的实时荧光定量PCR方法和病毒半数组织感染量(TCID50)方法测定并比较猪JEV在BHK-21细胞中的增殖规律。结果表明：建立的猪JEV实时荧光定量PCR检测方法的扩增效率为99.3%,敏感性、特异性和重复性良好，检测敏感性可以达到1.0×101 copies/μL;TCID50方法测定的细胞悬液和细胞培养上清液中的病毒含量分别为1.0×105.44 TCID50/0.1 mL和1.0×104.86 TCID50/0.1 mL,实时荧光定量方法测定的病毒粒子数量分别为1.0×107.50 copies/μL和1.0×105.60 copies/μL,两种方法在细胞悬液中...  相似文献   

2.
为建立猪繁殖与呼吸综合征病毒(PRRSV)SYBR GreenⅠ实时荧光定量PCR检测方法,采用RT-PCR扩增PRRSV N基因266bp片段,并克隆到pMD18-T载体中,以纯化的重组质粒为模板进行荧光定量PCR扩增,建立了PRRSV荧光定量PCR检测方法,该方法检测灵敏度可达1.3×101拷贝/μL,与猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪日本脑炎病毒(JEV)均不发生交叉反应,具有很好的特异性和重复性。结果表明,建立的PRRSV实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点,可用于临床猪繁殖与呼吸综合征病毒病(PRRS)的诊断。  相似文献   

3.
为了建立猪圆环病毒2型(PCV2)SYBR GreenⅠ荧光定量PCR检测方法,采用PCR扩增PCV2 ORF2基因242 bp片段,并克隆入pMD18-T载体中,以纯化的重组质粒为模板作荧光定量PCR扩增,建立了PCV2荧光定量PCR检测方法,该方法检测灵敏度可达1.2×10~1拷贝/μL,与猪圆环病毒1型(PCV1)、猪瘟病毒(CSFV)、猪蓝耳病病毒(PRRSV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、乙型脑炎病毒(JEV)核酸均不发生扩增反应,具有很好的特异性和重复性。结果表明:建立的PCV2实时荧光定量PCR检测方法具有特异、敏感、快速、定量、重复性好等优点,可用于临床PCV2的检测。  相似文献   

4.
荧光定量RT-PCR检测猪日本脑炎病毒   总被引：1，自引：0，他引：1  
通过RT-PCR方法克隆了日本脑炎病毒(JEV)N基因片段序列,构建重组质粒作为标准阳性模板,同时根据GenBank中的靶序列设计了荧光定量R-PCR的1对引物和1条TaqMan探针.通过条件优化,以定量的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JEV检测的荧光定量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和1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为17.33、20.52和23.90,变异系数分别为0.54%、0.74%和0.53%,均小于5%.对阳性组织病料的检测表明该方法的灵敏度高出常规PCR,与套式PCR(nested-PCR,nPCR)具有相近的灵敏度.  相似文献   

5.
为了建立猪圆环病毒2型（PCV2）SYBR Green I荧光定量PCR检测方法,采用PCR扩增PCV2 ORF2基因242 bp片段,并克隆入pMD18-T载体中,以纯化的重组质粒为模板作荧光定量PCR扩增,建立了PCV2荧光定量PCR检测方法,该方法检测灵敏度可达1.2×101拷贝/μL,与猪圆环病毒1型（PCV1）、猪瘟病毒（CSFV）、猪蓝耳病病毒（PRRSV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、乙型脑炎病毒（JEV）核酸均不发生扩增反应,具有很好的特异性和重复性。结果表明：建立的PCV2实时荧光定量PCR检测方法具有特异、敏感、快速、定量、重复性好等优点,可用于临床PCV2的检测。  相似文献   

6.
用PCR方法扩增出鸡减蛋综合征病毒（EDSV）Hexon基因保守片段,经琼脂糖凝胶电泳及序列测定分析扩增产物的特异性。以构建的阳性重组质粒作为标准品,建立SYBR GreenⅠ实时荧光定量PCR反应的扩增曲线和溶解曲线,并绘制标准曲线。结果表明,建立的EDSV荧光定量PCR标准曲线Ct值与1×10^1～1×10^6拷贝/μL的基因拷贝数呈现良好线性关系,灵敏度可达10拷贝,且特异性及重复性良好;说明本试验建立的SYBR GreenⅠ实时荧光定量PCR检测方法可用于EDSV的诊断及病原的定量分析。  相似文献   

7.
为建立同时快速鉴别检测羊痘病毒属病毒(CaPV)的方法,本研究设计了一对针对CaPV的通用引物和3条特异性的TaqMan MGB探针,并对其反应条件进行优化,建立了单管同时鉴别检测3种病毒的多重TaqMan MGB荧光定量PCR方法。结果显示,该多重荧光定量PCR方法仅对牛结节性皮肤病病毒(LSDV)、山羊痘病毒(GTPV)和绵羊痘病毒(SPPV)有特异性扩增,而对牛痘病毒等其它相关病毒核酸无扩增。在10~2拷贝/μL～107拷贝/μL浓度范围内有良好的线性关系;对LSDV、 GTPV、SPPV的最低检测限分别为14.8拷贝/μL、32.9拷贝/μL、26.7拷贝/μL。标准曲线相关系数(R2)均为0.999。批内及批间变异系数均小于1.5%。应用本研究建立的方法和OIE陆生动物手册推荐普通PCR方法分别对185份临床样品和67份模拟样品进行检测,该方法检出率比普通PCR方法高约1.6%。本研究建立的CaPV多重TaqMan-MGB荧光定量PCR方法具有特异性强、敏感性高、重复性好、操作简便快速等优点,适用于CaPV临床样品的快速鉴别检测。  相似文献   

8.
为建立一种快速、敏感检测水貂阿留申病毒(AMDV)的方法,本研究根据AMDV的VP2基因保守区设计特异性引物和MGB探针,并优化检测反应条件,建立了AMDV的TaqMan荧光定量PCR检测方法。结果显示,以重组质粒为标准品建立的标准曲线在1.0×10~2拷贝/μL~1.0×10~8拷贝/μL内具有良好的线性关系,相关系数(R~2)为0.998。该方法仅对AMDV的靶基因扩增呈阳性,而对猪细小病毒、犬细小病毒、水貂肠炎细小病毒等相关病毒检测结果均为阴性,特异性良好。该方法对AMDV检测的灵敏度为10拷贝/μL,为普通PCR灵敏度的100倍;组内和组间重复性试验表明该方法的组内和组间变异系数均小于2%,具有良好的重复性。应用建立方法和普通PCR方法分别对40份疑似临床组织病料样品进行检测,该方法检出率比普通PCR高约7.5%。本研究结果表明建立的AMDV TaqMan-MGB荧光定量PCR方法灵敏、快速、特异性强、重复性好,适用于对AMDV的快速定量检测。  相似文献   

9.
为了了解猪瘟病毒(CSFV)在PK-15细胞上的增殖规律。根据GenBank中CSFV Shimen毒株的NS5A基因保守区域序列,设计1对特异性引物,以CSFV总RNA为反转录模板,经优化反应条件,建立了基于实时荧光定量PCR技术的CSFV检测方法,并对其进行了敏感性、特异性、重复性试验,结果显示,该方法重复性好、特异性强,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1 TCID_(50 )/mL。利用所建立的方法研究CSFV在PK-15细胞上清中的增殖动态,并与TCID_(50)方法绘制的复制动态曲线进行比较。结果显示,两种方法测定的CSFV在PK-15细胞上的复制动态具有一定的平行关系,该方法为研究猪瘟病毒的致病机理及利用体外细胞培养毒生产疫苗提供了试验依据。  相似文献   

10.
猪流感病毒实时荧光定量RT-PCR检测方法的建立   总被引：2，自引：1，他引：1  
本研究选取猪流感病毒(swine influenza virus,SIV)的NP基因序列设计引物和探针,建立了检测SIV的TaqMan实时荧光定量PCR方法。以梯度稀释的含有SIV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度。2.0×108至2.0×102拷贝/μL 7个数量级的范围内定量PCR有"S"型扩增曲线,检测灵敏度为20个拷贝/μL。根据病毒拷贝数与Ct值的关系绘制了标准曲线。该方法具有特异性,对猪瘟病毒、伪狂犬病病毒、口蹄疫病毒和猪繁殖与呼吸综合征病毒核酸都没有扩增反应。本研究建立的实时定量PCR方法,灵敏度高、特异性好,可以进行定量分析,在猪流感的快速检测上具有重要意义。  相似文献   

11.
1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.  相似文献   

12.
Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.  相似文献   

13.
Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.  相似文献   

14.
Tropical Animal Health and Production -  相似文献   

15.
The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.  相似文献   

16.
The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.  相似文献   

17.
Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.  相似文献   

18.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
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19.
An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.  相似文献   

20.
Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein  相似文献   

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