检测流感病毒的商业PCR试剂盒比对验证

利用8种不同流感病毒株及其核酸,结合自然感染组织样品与人工混合样,验证比较了8种商业PCR试剂盒的质量参数。结果表明,3号荧光PCR试剂盒漏检1株外,其他试剂盒均可以检测8种毒株,对其他类似病毒如猪繁殖与呼吸综合征病毒、猪伪狂犬病毒、猪传染性胃肠炎病毒、猪细小病毒和猪环状病毒核酸等无交叉反应,最高可检测到10-7稀释的目标核酸,重复性与再现性均为理想,在有效期内检测结果没有发生变化。     

动物布鲁氏菌病实验室检测技术研究进展

布鲁氏菌病是由布鲁氏菌引起的人畜共患传染病,对我国畜牧业生产和公共卫生安全造成严重的威胁。科学准确诊断是布鲁氏菌病防控的重要技术依据,然而布鲁氏菌病的检测和确诊一直以来都并非易事,常常需要通过多种检测结果的综合分析,因此发展敏感性高、特异性好的布鲁氏菌的检测技术十分重要。总结并分析了近年来布鲁氏菌检测技术基础研究和临床应用研究方面的最新进展,结合国家动物布鲁氏菌病参考实验室工作经验,对各类检测方法的优缺点和适用范围进行了分析,旨在为动物布病诊断技术的选择和推广应用提供理论和实践依据,为动物布鲁氏菌诊断技术的研究提供方向性思考。     

Advances in laboratory diagnosis of parasitic infections of sheep

Parasitic infections constitute an important group of diseases in sheep concerning the health status, welfare and productivity. On a global scale, there are considerable differences concerning the epidemiological situation with respect of the various parasite species. However, there are also numerous species, which occur on all continents and, potentially, in every country. Accordingly, the present review aims to providing an overview about the recent developments in methods and technologies for the laboratory diagnosis of parasite infections in sheep. Following in principle a systematic order the review encompasses publications addressing the diagnosis of helminthes (i.e., trematodes, cestodes and nematodes) and arthropod species. New approaches using conventional (e.g., microscopic), immunological and molecular techniques are being considered. The diagnosis of anthelmintic resistance is highlighted separately, due to its significant importance. The review ends with an outlook into the future by discussing most recent technological advances, which might become of use for the diagnosis of parasite infections in sheep in the future.     

Validation of thromboelastometry in horses

Background: Thromboelastometry is used for identifying or monitoring coagulation abnormalities. It has been validated in several species but not in horses and the characteristics of the equine thromboelastogram have not yet been detailed. Objectives: The purpose of this study was to validate a thromboelastometer to be used with equine blood and to define the normal equine thromboelastogram. Methods: A Rotem-gamma thromboelastometer (Pentapharm GmbH, Munich, Germany) was used on 38 citrated blood samples to investigate native coagulation, the intrinsic and extrinsic pathways, the function of fibrinogen (largely dependent on its concentration), and the presence of fibrinolysis. Using classic validation approaches, we evaluated the imprecision of the method and the influence of hemolysis and storage time and temperature. The normal thromboelastogram was defined in both saddle and racing horses (the latter sampled before and after the race). Results: For imprecision tests, the analytical variations were <10%. The equine thromboelastogram had a pattern similar to those of other species, but the intrinsic and extrinsic pathways were less and more efficient, respectively. Reference intervals in racing horses, especially after exercise, were different from those of saddle horses, most likely due to a higher RBC mass. Coagulability decreased in hemolyzed samples and significant changes were found between nonrefrigerated and refrigerated blood samples stored for 20 hours. Conclusions: The Rotem-gamma thromboelastometer is a precise instrument for use with equine blood samples. The equine thromboelastogram is similar to that of other species, but reference intervals vary with aptitude and exercise. Hemolysis and refrigeration alter thromboelastometric results.     

Comparison of direct immunofluorescence, immunoassays, and fecal flotation for detection of Cryptosporidium spp. and Giardia spp. in naturally exposed cats in 4 Northern California animal shelters

BACKGROUND: Giardia spp. and Cryptosporidium spp. are common intestinal protozoan parasites in domestic cats. Few studies have critically evaluated the performance characteristics of commercially available immunoassays for detection of these organisms in the cat. HYPOTHESIS: Human-based immunoassays are suboptimal for the detection of Giardia spp. and Cryptosporidium spp. in cats. ANIMALS: Three-hundred-and-forty-four cats with diarrheic and nondiarrheic fecal specimens at 4 northern California animal shelters. METHODS: A fecal specimen was collected from each cat in a case-controlled fashion. Fecal specimens were tested for Giardia spp. and Cryptosporidium spp. by using centrifugation flotation and 5 commercially available immunoassays (SNAP Giardia, ProSpecT Giardia Microplate Assay, ProSpecT Cryptosporidium Microplate Assay, ImmunoCard STAT! Cryptosporidium/ Giardia Rapid Assay, and Xpect Giardia/Cryptosporidium). Results were compared with a reference standard, the MeriFluor direct immunofluorescence assay. RESULTS: Overall prevalences of Giardia spp. and Cryptosporidium spp. were 9.8 and 4.7%, respectively. The ProSpecT Microplate Assay had the highest sensitivities and specificities for Giardia spp. (91.2 and 99.4%) and Cryptosporidum spp. (71.4 and 96.7%), respectively. The SNAP Giardia antigen assay was easier to use and equally sensitive (85.3%) and specific (100%) to fecal flotation. CONCLUSIONS AND CLINICAL IMPORTANCE: Caution should be exercised when using human-based immunoassays for the diagnosis of Giardia and Cryptosporidium spp. in cats. Fecal flotation remains a useful method for detection of Giardia spp., can be used to detect other parasites, and has a sensitivity of 97.8% for detection of Giardia spp. when combined with the SNAP Giardia immunoassay.     

Validation of data collected in the Danish Veterinary Cancer Registry

The Danish Veterinary Cancer Registry (DVCR) was established in 2005 and prospectively collects information about neoplasms in Danish dogs and cats.
The present study evaluated the agreement between veterinary practice records and electronic submissions made to the DVCR from May 2005 through June 2008. The variables compared were gender, breed, diagnosis, date of birth, death and diagnosis, localization and biological behaviour of the neoplasms.
Agreement of 95% between DVCR and the original data were considered acceptable with regard to use of data in studies. Recorded proportions of agreement were: (1) breed: 97.4%; (2) diagnosis: 95.6%; (3) location: 95.6%; (4) biological behaviour: 93.0%; (5) gender: 92.5%; (6) date of death: 91.5%; (7) date of diagnosis: 80.1%; (8) date of birth: 76.7%.
All variables except date of death, birth, and diagnosis met the required 95% agreement limit. Data on dates of birth, death and diagnosis were considered less suitable for further studies.     

Quantitative correlation of parasitological and serological techniques for the diagnosis of Trypanosoma congolense infection in cattle

Summary

A comparison was made between serological and parasitological techniques for the diagnosis of bovine trypanosomiasis in Zambia. Overall sero‐prevalence rates as determined by IFAT and ELISA were respectively 2.7‐fold and 2.9‐fold greater then the percentage of samples found positive with the dark ground/phase contrast buffy coat technique (DG). The results obtained by the two serological techniques were found to be closely correlated (94.2% agreement) and titres obtained by ELISA tended to be slightly higher than those obtained by IFAT. Linear regression analysis of the results obtained by the IFAT and DG techniques revealed a highly significant correlation. This finding would permit the use of only one of the techniques in an epidemiological survey and to extrapolate the results from the regression line.     

Quantitative correlation of parasitological and serological techniques for the diagnosis of Trypanosoma congolense infection in cattle

A comparison was made between serological and parasitological techniques for the diagnosis of bovine trypanosomiasis in Zambia. Overall sero-prevalence rates as determined by IFAT and ELISA were respectively 2.7-fold and 2.9-fold greater then the percentage of samples found positive with the dark ground/phase contrast buffy coat technique (DG). The results obtained by the two serological techniques were found to be closely correlated (94.2%) agreement) and titres obtained by ELISA tended to be slightly higher than those obtained by IFAT. Linear regression analysis of the results obtained by the IFAT and DG techniques revealed a highly significant correlation. This finding would permit the use of only one of the techniques in an epidemiological survey and to extrapolate the results from the regression line.     

Quality control validation in veterinary laboratories

Quality control (QC) validation is used to determine: 1) whether statistical QC procedures are appropriate for detecting medically important errors; and 2) the equality of performance required by different laboratory tests. QC validation is well documented in the medical literature, but we are unaware of studies addressing its application, problems or unique differences in veterinary laboratories. We applied QC validation to automated hematology and biochemistry analyses in our laboratories, with goals of >/= 90% probability of error detection and 相似文献   

变性与非变性PAGE在检测家蚕微卫星PCR产物中差异

利用公布的家蚕基因组序列，设计特定的家蚕微卫星引物，对8个不同品种家蚕的微卫星位点进行PCR扩增，所得PCR产物经变性与非变性聚丙烯凝胶（PAGE）分离，银染后发现在非变性PAGE上，条带较粗且模糊，非特异带较多，导致读带误差增大；而在变陛PAGE上，所得条带较细且比较清晰，非特异带明显减少，有利于降低统计误差。     

Validation of two ELISA assays for total ghrelin measurement in dogs

The aim of this study was to validate two commercially available ELISA assays for total ghrelin measurement in dogs: one canine-specific and one originally designed for measuring human ghrelin. The two assays showed intra-assay coefficient of variations (CVs) lower than 10%, while the inter-assay CVs exceeded the 15% limit. Sample dilutions resulted in linear regression equations with correlation coefficients close to 1. In order to compare methods and verify ability of the ghrelin assays to differentiate between low and high levels, ghrelin concentrations were measured in plasma samples obtained before and at different times after glucose administration in five Beagle dogs. A statistically significant changes in ghrelin after glucose administration was recorded only with assay B. In conclusion, the human ELISA validated in this study showed a good intra-assay precision, accuracy, and when applied to the glucose injection study, was better in distinguishing high and low canine ghrelin levels than the canine ELISA assay.     

PCR技术在猪圆环病毒2型检测中的应用

参照Gen Bank中已发表的猪圆环病毒2型(PCV-2)ORF2基因序列,设计合成1对引物,建立了检测猪场疑似病例临床样本中PCV-2的PCR方法。结果显示,PCV-2的ORF2扩增产物经琼脂糖凝胶电泳,呈现1条630 bp的特异性条带,与预期目的片段大小一致。应用该方法对安徽省3个猪场的30份病料进行了PCV-2检测,结果有9份检测出PCV-2,阳性率为30%。结果表明,该试验建立的PCR方法,是一种快速、灵敏、高效的PCV-2检测技术。     

国外猪瘟病毒实验室诊断、流行病学以及标记疫苗研究进展

常用于检测猪瘟病毒的实验室诊断方法是免疫组化和病毒分离培养.随着分子生物学的发展,先后建立了一系列检测病毒蛋白的ELISA方法以及病毒基因组的RT-PCR方法.标记疫苗是目前猪瘟疫苗发展的趋势.目前已经完成E2亚单位标记疫苗及重组活疫苗效率的测定,均能显著减少疫苗免疫猪群中猪瘟病毒的水平及垂直传播,同时通过血清学方法能区分疫苗免疫猪与自然感染猪.标记疫苗的使用对于猪瘟的控制和消灭具有十分重要的意义.     

浅谈SoC模块级与系统级的验证

SoC芯片通常包含有复杂的数据通路,对复杂的数据通路的验证非常具有挑战性。无线通信SoC中包括很多功能模块,为了验证其复杂的数据通路的正确性,需要对每个功能模块进行模块级的验证。由于SoC验证已经成为整个流程中的重心,所以努力研发新的验证方法及设备,不断完善SoC验证计划和平台是当前的主要任务。以SD控制器和芯片系统（SoC）为例,探讨SoC模块级和系统级的验证,先确定验证策略并编写测试计划,再创建测试平台,最后对复杂的数据进行测试。     

Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs

Six surra negative piglets (6-week-old) were infected with Trypanosoma evansi and two uninfected piglets were used as negative controls. Detection performances of various diagnostic tests (LAMP, PCR and parasitological tests) were compared by analysing blood samples collected weekly over a period of 11 weeks. With a two by two analysis without a gold standard, all methods were 100% specific. MI had the highest sensitivity of 65%, while LAMP, PCR, MHCT and TBS had sensitivities of 45, 33, 38 and 24%, respectively. However, when the analysis was done using MI as a gold standard, the sensitivity of MHCT was the highest at 53% followed by LAMP, PCR and TBS at 49, 44 and 35%, respectively. All methods gave high specificity above 60%. This study validates LAMP as an alternative method for the diagnosis of surra.     

Validation of the QTL on SSC4 for meat and carcass quality traits in a commercial crossbred pig population

Porcine chromosome 4 harbours many quantitative trait loci (QTL) affecting meat quality, fatness and carcass composition traits, detected in resource pig populations previously. However, prior to selection in commercial breeds, QTL identified in an intercross between divergent breeds require confirmation, so that they can be segregated. Consequently, the objective of this study was to validate several QTL on porcine chromosome 4 responsible for meat and carcass quality traits. The experimental population consisted of 14 crossbred paternal half-sib families. The region of investigation was the q arm of SSC4 flanked by the markers S0073 and S0813. Regression analysis resulted in the validation of three QTL within the interval: Minolta a * loin, back fat thickness and the weight of trimmed ham. The results were additionally confirmed by factor analysis. Candidate genes were proposed for meat colour, which was the most evident QTL validated in this study.     

High-resolution melt curve analysis: A real-time based multipurpose approach for diagnosis and epidemiological investigations of parasitic infections

BackgroundReal-time PCR coupled with high resolution melting curve analysis is a practical technique that could be employed in multipurpose studies. During the recent decade, this technique has been practiced for different targets, worldwide.MethodsIn the current study three major database centers consisted of PubMed, Scopus and Web of Science were searched until Aug 2019 for applications of HRM real-time PCR in parasitology studies using terms: “Parasite” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”, “Protozoan parasites” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”, “Helminth” AND “HRM real-time PCR” OR “High Resolution Melting curve analysis” OR “Real-time PCR”.ResultsTotally, 83 papers met our criteria and were included in our study. This method was more frequently used for protozoan parasites (52/83; 62.65%), while lower (31/83; 37.35%) studies were incorporated on helminths parasites. Furthermore, Plasmodium spp., and Leishmania spp., were the most prevalent protozoan parasites, and Taenia spp., and filers were the most frequent helminths that were studied by HRM real-time PCR.ConclusionHRM real-time PCR is a sensitive, flexible and cost-effective method that could be used for multipurpose studies.     

Validation of an immunoturbidimetric D-dimer assay in canine citrated plasma

Abstract: D-dimer is a neoantigen formed when thrombin initiates the transformation of fibrinogen to fibrin; it is derived from plasmin digestion of cross-linked fibrin. In human medicine, the usefulness of this analyte in diagnosing disseminated intravascular coagulation (DIC) has been assessed in patients fulfilling the clinical and laboratory requirements for this disorder. In canine medicine, the use of D-dimer is relatively new. Detailed studies are needed to understand the relationship between D-dimer concentration in plasma and DIC status in dogs. We validated a D-dimer immunoturbidimetric assay (Tina-quant [a] D-Dimer, Boehringer Mannheim) in canine citrated plasma samples. Intra-assay and interassay variability (coefficient of variation) was 5.63% and 8.82%, respectively. The assay was linear, using 2 samples with low and high D-dimer concentrations (r = .996 and .998). Accuracy was 102.2% and 95.7% based on a recovery study in which 2 samples were assessed. Reference values for D-dimer were established using 70 healthy dogs that were assessed clinically and evaluated on the basis of a complete laboratory workup. The reference range was set between 0.02 and 0.28 μg/mL (chi-square test for normal distribution, P > .05).     

Validation of the Nova CRT8 for the measurement of ionized magnesium in feline serum

Background: Evaluation of serum magnesium (Mg) concentration is becoming important in human and veterinary critical care medicine. An ion‐selective electrode can measure the physiologically active ionized fraction. Objectives: The purpose of this study was to validate an ion‐specific electrode analyzer and assay for measuring ionized Mg in feline serum and to determine a reference interval for this analyte in cats. Methods: Venous blood samples were collected anaerobically from clinically healthy cats, and the serum was used to validate the analyzer and assay. This included investigating the stability of samples stored at different temperatures, intra‐ and interassay precision, linearity, analytical sensitivity, and potential interferences from bilirubin, lipemia, hemoglobin, or serum separator tubes. A reference interval was calculated. Results: Serum samples evaluated for ionized Mg concentrations can be stored at 20°C for ≤24 hours, at 4°C for ≤72 hours, and at ?20°C for ≤4 weeks, when samples are minimally exposed to air. Intra‐ and interassay precisions had coefficients of variation (CVs) of 1.23% and 2.02%, respectively. There was good linearity using serum (r= .998; y=?0.0057 + 1.0256x) and manufacturer‐supplied aqueous solutions and quality control materials (r= .999; y= 0.0110 + 0.9213x). Apparent analytical sensitivity was at least 0.015 mmol/L. Mean recovery was good for ionized Mg in samples with ≤1+ icterus (104%), 4+ lipemia (99.3%) and 1–4+ hemolysis (98.6%). There was no significant difference (P= .52) in ionized Mg concentrations in serum collected in tubes containing no additives compared with serum collected in glass separator tubes. The serum ionized Mg reference interval was 0.47–0.63 mmol/L (n = 40). Conclusions: The Nova CRT8 analyzer and assay provide a precise and reliable method of measuring ionized Mg concentration in feline serum. Strict adherence to sampling techniques, handling, and storage are necessary for reliable results.