RT—PCR技术常见问题的分析

聚合酶链式反应（Polimerasechainreaction，简称PCR技术）具有特异性强、灵敏度高、简便快速、对原始材料质量要求低的特点，为兽医分子生物技术实验室中的首选方法之一。但因该技术高度的灵敏性容易导致交叉扩增反应，出现假阳性结果等，现就其常见问题作一探讨。１PCR技术常出现的几个问题１．１假阳性实验中设立的阴性对照出现阳性结果，则实验检测结果可能有假阳性。造成假阳性的原因主要包括监测样品污染、监测试剂污染、扩增产物污染等。１．２假阴性实验中设置的阳性对照未能扩增成阳性结果，则实验可能有假阴性结果。但应注意，…     

猪蓝耳病抗体试剂盒出现假阳性的确诊思路

猪蓝耳病（PRRS）是影响养猪行业的一个重要的疾病。对于一个核心场或者公猪站而言,维持猪蓝耳病双阴性,做好猪蓝耳病的监测工作是及其重要的。猪蓝耳病抗体检测是猪蓝耳病双阴性场评估猪群是否感染猪蓝耳病野毒常用的方式之一。但是所有商品化的试剂盒的敏感性和特异性并不是100%准确的。通过对3个猪蓝耳病双阴性猪场的猪只进行猪蓝耳病抗体检测,检测结果分别出现2.17%、100%、1.6%的阳性样本。对于异常结果除实验室进行复检和临床诊断外,还通过抗体变化的监测和免疫荧光的检测来验证检测结果的可靠性,经过验证得知这3个猪场出现的2.17%、100%、1.6%的阳性结果均为假阳性。     

蛋鸡问题血清调查及原因分析

某蛋鸡场从2009年8月开始陆续发生送检血清不同程度浑浊、高血脂、胶冻状等问题，尤其以开产前后更为严重，比例可高达到90％以上，血清问题给常规血清学检测带来干扰；导致假阳性、假阴性结果出现。血清状态的好坏直接反映鸡群的健康状况，为分析造成问题血清的原因，具体从以下几个方面进行了检测。     

影响“瘦肉精”残留快速检测假阳性的原因分析

近年来,随着我国对食品安全的进一步重视,"瘦肉精"的监督监测力度日益加大。然而在"瘦肉精"快速检测过程中,由于试剂盒及检测方法本身的缺陷,常常会出现一些假阳性。笔者在实验过程中曾多次遇到快速检测呈阳性,而通过色谱法确证又呈阴性的情况。鉴于此,现就影响"瘦肉精"快速检测假阳性的原因总结如下。     

ELISA检测中出现假阴性探讨

本文应用酶免疫试验（ＥＬＩＳＡ）、反向间接血凝试验（ＲＩＨＡ）和中和试验（ＮＴ）三种方法对６份疑似ＨＢＶ感染的待测血清进行测定，借此探讨ＥＬＩＳＡ检测中出现假阴性现象，认为假阴性是由检测前滞现象引起。     

PCR假阳性的原因分析及控制措施

近二十年来,PCR作为一种重要的分子生物学技术,广泛应用于动物疫病的病原学、诊断方法、基因工程疫苗研究、检验检疫、疫病诊断、疫情监测及流行病学调查等动物疫病防控实际工作。如何控制好PCR检测的质量,减少假阳性,一直是研究者普遍关注的问题。本文分析了PCR假阳性形成的原因,提出预防及控制措施,为促进实验室PCR实践工作提供参考。     

非洲猪瘟病毒荧光PCR检测假阳性的原因与预防措施

非洲猪瘟病毒(African swine fever virus,ASFV)荧光PCR检测是防控非洲猪瘟(African swine fever,ASF)的关键环节。江苏省动物疫病预防控制中心对两家具备ASF检测资质的实验室,进行了阳性样品复核,发现检测结果存在假阳性。为避免实验室检测中出现检测失误,本文分析了导致ASFV检测假阳性结果的原因:实验室检测功能区域设置以及人员、物品流向不合理;检测人员操作不规范、不熟练,未在独立的生物安全柜中进行样品提取与试剂配制;实验室通风不良、清洁消毒不彻底,样品或提取模板间产生交叉污染,PCR试剂、产物、阳性质粒发生污染。为此,从实验室结构、环境、操作等方面提出控制建议:严格实验室功能分区与管理,规范各种试验操作与流程,加强实验室通风和消毒,控制试验中产生PCR污染的相关因素;同时省级实验室要加强对基层实验室的培训、指导和监督,使其在ASF防控中发挥应有的关键作用。本文为从事ASF等重大动物疫病PCR检测的实验室,在提高检测结果准确性和可靠性方面提供了参考。     

皮内变态反应试验检测奶牛结核病的假阳性率初步测定及对策

笔者用牛型提纯结核菌素皮内变态反应试验对5425头奶牛进行结核病监测检测,然后间隔42d对前者检测出的阳性牛或可疑牛再次进行结核病检测,比较前后两次的阳性牛或可疑牛符合率.结果显示,前次应用皮内变态反应试验共检测出结核病阳性牛或可疑牛共计142头;再次检测呈阳性或可疑的142头奶牛中,结果仍为阳性牛121头.结果表明,皮内变态反应试验存在一定的假阳性,假阳性率约为14.8%(21/142),建议用皮内变态反试验监测检测奶牛结核病时,对检测出的阳性牛开展1次复核,以提高结核病检测的准确性和降低养殖业主的经济损失.     

酶联免疫吸附分析法检测克仑特罗时假阳性问题初探

以麻黄碱作为实验药物 ,对ELISA法检测克仑特罗的非特异性反应作了初步探讨。实验结果表明 ,结构中含有苯乙胺醇基团的麻黄碱能导致假阳性结果 ,并提示其它结构中含有苯乙胺醇基团的药物有可能导致假阳性结果     

中药成分辛弗林对胶体金免疫层析法检测β-受体激动剂的影响

旨在探究陈皮、青皮、木瓜、厚朴、红景天等5种中药材提取液以及饲喂了陈皮和青皮的猪尿液胶体金免疫层析法检测结果呈阳性的原因。建立测定猪尿及中药提取物中辛弗林的LC-MS/MS检测方法，测定陈皮、青皮的提取物和饲喂陈皮、青皮后猪尿中的辛弗林浓度。用小鼠肝微粒体孵育CGIA检测呈阳性的陈皮、青皮、木瓜和厚朴4味中药提取物。饲喂陈皮和青皮后的猪尿液检出辛弗林，浓度分别为1.36和1.65 μg·mL^-1；在陈皮和青皮的提取复溶液中检出辛弗林，浓度分别为132.6和312.7 μg·mL^-1。厚朴和木瓜两种中药提取物经过肝微粒体孵育后，在2~5 h逐渐由CGIA检测阳性转为阴性，陈皮和青皮孵育后，0~6 h的结果均为CGIA检测阳性；阴性中药组CGIA检测均呈阴性，而添加辛弗林的阳性对照组0~6 h CGIA检测均呈阳性。猪较大剂量使用陈皮和青皮会引起猪尿β-受体激动剂CGIA检测出现假阳性，该假阳性现象跟中药成分辛弗林有关，体外试验中辛弗林不易被小鼠肝微粒体代谢。     

Examination of milk samples from experimentally infected heifers by an N-acetyl-β-D-glucosaminidase test and an antigen-capture elisa

Milk samples collected from normal and experimentally infected quarters of five heifers throughout their first lactation were examined by bacterial culture, milk cell count (MCC), N-acetyl-beta-D-glucosaminidase (NAGase) test and a monoclonal antibody based antigen-capture ELISA. The results were analysed according to the presence (mastitis positive) or absence (mastitis negative) of bacteria on culture, and the numbers of false negative and false positive results of the other three tests defined in relation to this. Similar numbers of false negative results were observed with the MCC (20), NAGase (18) and ELISA (13). False positive results due to the physiological factors present in early lactation were evident in the MCC, prominent in the NAGase test and absent from the ELISA. The major difference in false positive results associated with experimental infection between the three tests was the more rapid return to negative values of the ELISA following resolution of infection, compared with MCC and NAGase.     

DOHUBLE-PHASE PARATHYROID SCINTIGRAPHY IN DOGS USING TECHNETIUM-99M-SESTAMIBI

The purpose of this study was to evaluate the utility of double-phase parathyroid scintigraphy using 99mTc-sestamibi for detecting and localizing hyperfunctioning parathyroid glands in hypercalcemic dogs. Fifteen hypercalcemic dogs that underwent parathyroid scintigraphy were included in this study: 3 dogs with hypercalcemia of malignancy, and 12 dogs with hyperfunctioning parathyroid tissue (parathyroid adenoma or parathyroid hyperplasia). The presence of parathyroid adenoma or parathyroid hyperplasia was documented by histopathologic examination. In 3 dogs with hypercalcemia of malignancy, parathyroid scintigraphy was negative for hyperfunctioning parathyroid tissue and the scans were classified as true negative. Parathyroid scintigraphy correctly identified the presence and location of hyperfunctioning parathyroid tissue in only 1 of 6 dogs with a parathyroid adenoma. False positive and false negative results occurred in dogs with parathyroid adenomas. Parathyroid scintigraphy failed to detect hyperfunctioning parathyroid tissue in 5 of 6 dogs with parathyroid hyperplasia and were classified as false negative. False positive results were obtained in the remaining dog with parathyroid hyperplasia. Sensitivity of parathyroid scintigraphy for detecting and localizing hyperfunctioning parathyroid tissue was 11%, specificity was 50%, and overall accuracy was 27%. Positive and negative predictive value were 25% and 27%, respectively. Sensitivity for detection of parathyroid adenomas was 25%, and sensitivity for detection of hyperplastic glands was 0 %. Results of this study indicate that double-phase parathyroid scintigraphy does not appear to have acceptable accuracy in detecting hyperfunctioning parathyroid glands in dogs. Due to the poor sensitivity and specificity of the technique in dogs, parathyroid scintigraphy is not recommended for definitive identification of abnormal parathyroid glands as the cause of hypercalcemia in dogs.     

感官鉴别真伪鹿鞭

通过对真伪鹿鞭感官真伪鉴定的研究 ,找出真伪鹿鞭的不同特点 ,并加以区分     

Evaluation of Fecal Elastase and Serum Cholecystokinin in Dogs with a False Positive Fecal Elastase Test

Background: An assay for the measurement of pancreatic elastase in dog feces has been introduced. Hypothesis/Objectives: The goal of this study was to evaluate the rate of false‐positive fecal‐elastase test results in dogs with suspected exocrine pancreatic insufficiency (EPI) and to assess serum cholecystokinin (CCK) concentrations in dogs with a false positive fecal elastase test result. Animals: Twenty‐six fecal and serum samples from dogs suspected of EPI, for which samples had been submitted to a commercial laboratory (Vet Med Labor) for analysis. Methods: Prospective study. Serum trypsin‐like immunoreactivity (TLI) was measured in 26 dogs with a decreased fecal elastase concentration of <10 μg/g feces. Serum CCK concentrations were measured in 21 of these dogs. Results: Of 26 dogs with a decreased fecal elastase concentration, 6 (23%) had serum TLI concentrations within or above the reference range. Serum CCK concentrations were significantly higher in dogs with a true positive fecal elastase test result (median: 1.1 pmol/L; range: 0.1–3.3 pmol/L) than in those with a false positive fecal elastase test result (median: 0.1 pmol/L; range: 0.1–0.9 pmol/L; P value = .0163). Conclusions and Clinical Importance: The rate of false positive fecal elastase test results was high in this group of dogs, suggesting that diagnosis of EPI must be confirmed by other means. The decreased CCK concentration in dogs with a false positive fecal elastase test result could suggest that false positive results are because of decreased stimulation of exocrine pancreatic function caused by other conditions.     

Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR)

Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was constructed by insertion of rpoB fragments from Rhodococcus equi into CEM-1P, which is a recombinant plasmid that includes a T. equigenitalis-specific sequence region. In CEM-PCR, the size of the PCR product from CEM-POS was clearly different from the true positive PCR product. In addition, CEM-POS retained high stability under convenient storage conditions of 4 degrees C. These results suggest CEM-POS to be a useful tool as a reaction control in routine CEM-PCR examinations.     

牛型结核菌素皮内变态反应试验和γ-干扰素ELISA试验检测奶牛结核病的比较研究

本研究采用PPD皮内变态反应试验和γ-干扰素ELISA试验,对甘肃省3个地区的1585头奶牛进行结核病检测.结果表明,PPD皮内变态反应共检出7份阳性样品;经γ-干扰素ELISA检测2份为阳性、其余5份为假阳性或禽型阳性,假阳性或禽型阳性样品再经细菌分离鉴定表现为阴性;PPD皮内变态反应检出的21份疑似样品再经γ-干扰素ELISA检测,表现为禽型阳性、假阳性或阴性;PPD皮内变态反应阴性样品经γ-干扰素ELISA试验检测,结果为阴性或禽型阳性.在检测奶牛结核病时,PPD皮内变态反应试验特异性较差,γ-干扰素ELISA试验结果与牛结核分枝杆菌细菌分离鉴定结果一致,而且该技术敏感性、特异性和鉴别假阳性均优于PPD皮内变态反应试验.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Fracture of the ischium in an eight‐year‐old Arabian gelding: A diagnostic challenge

Proximal hindlimb lameness remains a diagnostic challenge despite modern imaging techniques. In the case described here, a fracture of the ischium produced false negative results on initial ultrasound and scintigraphy examinations, despite a 14 day delay from onset of clinical signs to the time of the nuclear bone scan. However, the history and clinical examination were strongly suggestive of a pelvic injury and this was only confirmed by the use of a novel radiographic technique. False negative findings from diagnostic imaging can, and do, occur, even with high sensitivity techniques such as nuclear bone scans used after an appropriate delay period. Therefore, equal weighting should be given to all facets of a lameness investigation, including the history and clinical examination, while remembering the limitations of any imaging modalities used.