乳牛布鲁氏菌病病原DNA快速检测技术的研究

在国内首次建立了乳牛原乳中布鲁氏菌外膜蛋白(OMP)25ku基因套式聚合酶链反应(nested—PCR)检测技术。结果显示，本方法的特异性好，只能扩增布鲁氏菌DNA，而不能扩增同样能引起乳牛流产的鹦鹉热衣原体、弓形虫、胎儿弯杆菌DNA；乳样的检测灵敏度达50CFU／mL，重复试验35次，可重复性和稳定性均十分理想；对4批331头凝集试验阳性(24份)或阴性(307份)乳牛的乳样用本方法检测，符合率为100％。在48h内即可获得诊断结果。本方法同样可用于血液或其他流产病料中布鲁氏菌DNA的检测。     

新疆地区绵羊布鲁氏菌omp25基因PCR扩增研究

根据GenBank发表的绵羊布鲁氏菌标准菌株(63/290)的omp25基因序列设计引物,以新疆地区绵羊布鲁氏菌(80/019)基因组DNA为模板,探索PCR反应的各种条件以确定最适PCR反应条件。结果扩增出清晰的omp25目的基因片段,为今后进行omp25的分子生物学特性的研究奠定基础。     

流产布鲁氏菌omp25基因的克隆与原核表达

用设计的1对特异性引物对流产布鲁氏菌2型CVCC70502株总DNA进行外膜蛋白基因omp25的PCR扩增，得到了1条完整的基因，大小为642bp；测序分析证明，它与国外报道的流产布鲁氏菌omp25基因的核苷酸序列完全一致。将该基因克隆到原核表达载体pGEX-6p-1中，经酶切、PCR扩增和测序分析，表明重组表达载体构建成功。将此重组质粒转化至宿主菌BL21（DE3）中，用IPTG进行诱导。结果证实，该基因可以在大肠杆菌中表达，表达产物为分子质量约50ku的融合蛋白，与理论推测的蛋白分子质量一致；Western—blotting试验证明，表达蛋白OMP25可被流产布鲁氏菌阳性血清所识别。     

布鲁氏菌病VirB8-PCR诊断试剂盒的特异性评价

使用VirB8-PCR诊断试剂盒对布鲁氏菌标准株、疫苗株及新疆分离株共7株布鲁氏菌的VirB8基因序列扩增、克隆并与Genbank中发表的VirB8基因序列(AF226278)进行比较和分析,发现:该基因非常保守,7株布鲁氏菌的同源性在99.2%以上,其中A菌与Rev1基因序列100%同源,B菌与Genbank发表的基因序列100%同源;牛种(544A、A19)和羊种(M5、Rev1)分别各自在相同部位发生了相同的碱基突变,因此VirB8基因有可能做为布鲁氏菌疫苗菌的鉴定分类基因。VirB8基因在布鲁氏菌中高度保守,可作为布鲁氏菌检测模板,VirB8-PCR诊断试剂盒特异、敏感,检测结果可靠。     

布鲁氏菌病的实验室诊断方法综述

本文通过对布鲁氏菌病的实验室诊断方法进行综述,比较了直接诊断方法和间接诊断方法的优缺点,并介绍了目前先进的实验室诊断技术.笔者认为在当前形势下,为有效监测地区或国家的布鲁氏菌病流行情况,减少经济损失,应有针对性地、有所侧重地选择实验室诊断方法,以保障试验结果与实际情况一致相符,从而有效地预防与控制布鲁氏菌病.     

布鲁氏菌病实验室诊断技术的研究进展

布鲁氏菌病是由布鲁氏菌引起的一种重要人兽共患病。近年来,我国人、畜间布鲁氏菌病呈高发态势,严重威胁畜牧业生产和人民身体健康。及时准确诊断对防治和净化布鲁氏菌病具有重要意义,本文主要从病原学和免疫学两个方面对布鲁氏菌实验室诊断技术进行综述。     

牛结核病PCR诊断试剂盒的检测试验

应用自制牛结核病PCR诊断试剂盒对贵州省某奶牛场共计150份血样、奶样标本中结核分枝杆菌进行检测,结果显示:PCR试剂盒对111份奶样标本进行检测,8份为阳性,阳性检出率7.2%;PCR试剂盒对39份血样标本的检测,12份为阳性,阳性检出率为30.76%。本试验共计检测贵阳附近某奶牛场1 200头份奶牛,先采用PPD检测牛结核病阳性牛和可疑牛,再用PCR法进行检测,PCR检出阳性牛共计20头份,阳性检出率为1.67%;PPD检出阳性牛24头份,检出率为2%。总之,PCR试剂盒在检测牛结核病不同标本中显示出快速、特异等优点,这为今后牛结核病的检测工作提供了一条新途径。     

内参PCR法在布鲁氏菌病诊断中的应用

本试验以布鲁氏菌致病力因子VirB7下游至VirB9上游基因序列为目的扩增片段,设计上、下游引物,优化血样、奶样中布鲁氏菌基因组DNA的提取方法,建立布鲁氏菌内参PCR(IR-PCR)检测方法,用于血液、奶液样本中布鲁氏菌的检测。结果显示,内参PCR法有效地降低了PCR法诊断布鲁氏菌的假阴性率,且检测结果与常规的布鲁氏菌的诊断方法(细菌培养分离鉴定、iELASA)一致,该方法不仅提高了常规布鲁氏菌诊断方法的效率及灵敏度,而且降低了其假阴性和假阳性率。对血样、奶样的检出量分别为35CFU/mL、350 CFU/mL,适合于血样、奶样中布鲁氏菌的检测。     

牛结核病PCR诊断试剂盒质量标准的研究

根据GenBank中的牛结核分枝杆菌IS6110的基因片段,设计了1对引物,通过对PCR反应条件进行优化,研制了用于检测牛结核病的PCR试剂盒．该试剂盒的质量标准是：敏感性为3．04pg／μL、条带为478bp、特异性为100％,-20℃至少可保存12个月,重复性良好。     

新疆达坂城区家畜布鲁氏菌病免疫效果评估

[目的]了解达坂城区实行家畜布鲁氏菌病疫苗免疫后的免疫效果和疫情流行情况,有针对性地开展家畜布鲁氏菌病防控工作,制定更加合理的预防对策和控制措施。[方法]对达坂城区5个乡镇17个村的牛羊养殖户,以问卷调查和访谈结合的形式开展调查,并对结果利用SSPS和EXCEL软件进行数据统计分析。[结果]达坂城区实施家畜布鲁氏菌病免疫后,牛羊流产率由12%下降到6%,下降显著(P0.001);对布鲁氏菌病的知晓情况,非感染人群高于感染人群,总体个人防护意识增强。[结论]开展家畜布鲁氏菌病免疫对降低畜间和人间布鲁氏菌病发病率具有明显效果。     

安氏隐孢子虫PCR诊断试剂盒的初步应用

运用首次研制的安氏隐孢子虫PCR诊断试剂盒对广东省4个奶牛场和河南省1个奶牛场共234份样品,进行了安氏隐孢子虫感染的实际检测,并与常规检测方法饱和蔗糖漂浮法、改良抗酸染色法进行了比较。本试剂盒的检出率比常规检测方法提高了2%～13%,显示该试剂盒具有特异、敏感等优点,对开展隐孢子虫病的鉴别诊断和分子流行病学调查具有重要的应用价值。     

PCR as a diagnostic tool for brucellosis

Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of human brucellosis or contamination of food products). In these cases, a genus-specific PCR assay is sufficient. Genus-specific assays tend to be simple, robust, and somewhat permissive of environmental influences. The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S–23S rRNA operon.

Other instances require identification of the Brucella species involved. For example, most government-sponsored brucellosis eradication programs include regulations that stipulate a species-specific response. For epidemiological trace back, strain-specific identification is helpful. Typically, differential PCR-based assays tend to be more complex and consequently more difficult to perform. Several strategies have been explored to differentiate among Brucella species and strains, including locus specific multiplexing (e.g. AMOS-PCR based on IS711), PCR-RFLP (e.g. the omp2 locus), arbitrary-primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in molecular diagnostics for Brucella including the development of procedures designed for the direct analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for improvement.     

奶牛瘤胃微生物及其代谢物与乳脂合成关键酶基因对乳脂率的影响

乳脂肪对于人类的健康有着重要的生理作用,乳脂率的高低决定牛乳的品质、风味和营养价值,所以乳脂率对牛乳品质十分关键.奶牛乳脂率的高低受到多种因素的影响,如何提高奶牛乳脂率一直是行业研究的热点.大量研究表明,瘤胃微生物及其代谢产物与乳脂合成关键酶基因对奶牛乳脂率的高低具有重要调控作用.文章就瘤胃微生物及其代谢物与乳脂合成关...     

淡水养殖鱼类主要细菌性传染病快速诊断的研究——鱼病诊断箱的应用试验

应用以葡萄球菌Ａ蛋白协同凝集试验（ＳＰＡ－ＣＯＡ）为主要检测手段的鱼病诊断箱,于１９９５￣１９９７年在吉林,辽宁等７省进行现场检测试验。结果,６１个渔场１１个品种的５１６尾病鱼有菌生长的２８５尾,经ＳＰＡ－ＣＯＡ鉴定阳性数为２３９尾,占有菌生长的８３．８６％,１２个渔场５个品种１２１６尾假定健康鱼有菌生长的为２８尾,经ＳＰＡ－ＣＯＡ鉴定阳性数为２２尾,占有菌生长的７８．５７％,认为本诊断箱适用于基     

Evaluation of the Usefulness of a PCR Assay Performed at a Clinical Laboratory for the Diagnosis of Respiratory Disease Induced by Equine Herpesvirus Type 1 in the Field

A PCR assay for the diagnosis of respiratory disease induced by equine herpesvirus type 1 (EHV-1) was performed at the clinical laboratory in the Racehorse Clinic of the Ritto Training Center of the Japan Racing Association from December 2007 to March 2008. The assay was performed without the trouble of contamination throughout the study and its turnaround time was approximately 6 hr. The PCR detection rates of EHV-1 among seroconverted horses were 22.2% for nasal swabs and 33.3% for blood samples. However, EHV-1 DNA was also detected in horses without seroconversion at a low rate. These results indicated that the PCR assay should be used as an adjunct method, but would help to make an early diagnosis of EHV-1 infection.     

奶牛场荧光定量PCR检测实验室建设实践与思考——以原生态牧业奶牛场实验室建设为例

荧光定量PCR技术用于病原微生物基因表达、基因组变异和多态性检测等,具有灵敏度高、特异性高、快捷、对样品要求低等优点,已广泛用于临床诊断和畜禽疫病诊断。本文以黑龙江原生态牧业奶牛场荧光定量PCR检测实验室建设为例,从设计规划、配套设备、人员配备、环境控制及存在问题解决五大方面进行论述,提出PCR实验室建设要根据奶牛场场地实际情况,规划适合PCR实验检测区域;根据PCR检测需求及奶牛场费用预算配置实验设备;根据奶牛场预计检测样品量配备检测人员及培养储备人员;在建立严格的操作规范基础上,严格执行实验分区管理及检测过程中消毒流程,避免实验过程中产生气溶胶污染环境。     

A field study evaluation of Petrifilm™ plates as a 24-h rapid diagnostic test for clinical mastitis on a dairy farm

Clinical mastitis is one of the most common and expensive diseases of dairy cattle. To make an informed treatment decision, it is important to know the causative pathogen. However, no detection of bacterial growth can be made in approximately 30% of all clinical cases of mastitis. Before selecting the treatment regimen, it is important to know whether the mastitis-causing pathogen (MCP) is Gram-positive or Gram-negative. The aim of this field study was to investigate whether using two 3M Petrifilm™ products on-farm (which conveys a higher degree of sample freshness but also bears a higher risk for contamination than working in a lab) as 24-h rapid diagnostic of clinical mastitis achieved results that were comparable to the conventional microbiological diagnostic method.     

Estimation of genetic parameters for heat stress,including dominance gene effects,on milk yield in Thai Holstein dairy cattle

Heat stress in tropical regions is a major cause that strongly negatively affects to milk production in dairy cattle. Genetic selection for dairy heat tolerance is powerful technique to improve genetic performance. Therefore, the current study aimed to estimate genetic parameters and investigate the threshold point of heat stress for milk yield. Data included 52 701 test‐day milk yield records for the first parity from 6247 Thai Holstein dairy cattle, covering the period 1990 to 2007. The random regression test day model with EM‐REML was used to estimate variance components, genetic parameters and milk production loss. A decline in milk production was found when temperature and humidity index (THI) exceeded a threshold of 74, also it was associated with the high percentage of Holstein genetics. All variance component estimates increased with THI. The estimate of heritability of test‐day milk yield was 0.231. Dominance variance as a proportion to additive variance (0.035) indicated that non‐additive effects might not be of concern for milk genetics studies in Thai Holstein cattle. Correlations between genetic and permanent environmental effects, for regular conditions and due to heat stress, were ? 0.223 and ? 0.521, respectively. The heritability and genetic correlations from this study show that simultaneous selection for milk production and heat tolerance is possible.