四川猪2型链球菌病的PCR检测

2005年7月中下旬,四川贵阳等地人、猪陆续发生一种发病惠、死亡快、高度散发的疾病,从病死猪组织中分离出19株链球菌,设计合成3对引物,分别对分离菌株进行了猪链球菌16SrRNA、CPS2J、MRP基因片段扩增,有15株菌3个基因的扩增结果均为阳性,表明此次疲情的病原为猪2型链球菌。     

动物油脂中DNA的提取及牛、羊源性成分的PCR检测

用异硫氰酸胍法提取鱼油、加入牛肉DNA的鱼油、加入山羊肉DNA的互油和牛油中的DNA。18S rDNA片段以及牛、羊源性成分的PCR扩增结果表明，建立的动物油脂DNA提取方法是可行的。用建立的DNA提取方法提取3份送检鱼油的DNA，牛、羊源性成分的PCR检测结果均为阴性。     

猪繁殖与呼吸综合征病毒和副猪嗜血杆菌混合感染的诊断

四川省某种猪场70日龄仔猪发生以发绀、呼吸困难、多发性关节炎、肺出血、实变及多发性浆膜炎为特征的传染病,发病率为100％,死亡率为90％以上,经实验室诊断为猪繁殖与呼吸综合征病毒和副猪嗜血杆菌混合感染。     

肠出血性大肠杆菌O157毒力及黏附相关基因的PCR检测和PCR—RFLP分析

为了了解大肠杆菌O157分离菌株携带毒力及黏附相关基因的情况及菌株的多态性.用聚合酶链式反应扩增stx1、stx2、eaeA、ehxA、EspA和Tccp基因.选用限制性内切酶HincⅡ和EcoRⅡ对分离的O157:H7菌株eaeA基因进行酶切,选用限制性内切酶HaeⅢ、HinfⅠ、EcoRⅡ和RsaⅠ对分离的O157:H7菌株chxA基因进行酶切,最后对这两个基因进行PCR-RFLP分析.结果,所有O157:H7菌株扩增出eaeA、ehxA、EspA和Tccp基因,但没有扩增出stx1和stx2基因;4株O157:H?菌株,均没有扩增出stx2、eaeA、ehxA、EspA和Tccp基因,且只有1株扩增出stx1基因.所有分离菌株的eaeA基因和ehxA基因选用限制性内切酶酶切之后所得酶切片段数目和大小与标准菌株的eaeA基因和ehxA基因选用限制性内切酶酶切之后所得酶切片段数目和大小相同.结果表明,由于所有菌株缺失stx2基因,其致病力相对较低,且在基因水平较为保守,多态性较为单一.     

Study on some molecular characterization of Babesia orientalis

The study on buffalo babesiosis indicated that its pathogen was different from other Babesia on many aspects such as morphology, transmission and pathogenicity. Therefore, it was named as a new species—Babesia orientalis. In order to prove the validity of this taxon, molecular taxonomic study on the pathogen was done in this experiment. The complete 18S rRNA gene sequence of B. orientalis was determined by PCR. It was sequenced and blasted. The results indicated that the classification of the parasite belonged to the genus Babesia. The 1700 bp complete sequence was compared with 15 other Babesia sp. available in GenBank. The data were analyzed and a phylogenetic tree was established. The results indicated that the hereditary distance of the parasite was close to that of Babesia sp. from South Africa and Babesia ovis, and the hereditary distance was far from Babesia bigemina and B. bovis.     

3株猪源非结核分支杆菌16 S rRNA基因序列分析

用PCR方法从3株猪源非结核分支杆菌中扩增16 S rRNA基因5′端片段并进行序列测定。用DNA分析软件将所获得的序列与GenBank中分支杆菌序列相比较,计算种间相似性,构建系统进化树,对菌株进行分类与鉴定。结果表明,3株猪源非结核分支杆菌中浅黄分支杆菌1株,偶发分支杆菌2株。非结核分支杆菌在猪中存在的现象应引起重视,对人畜的影响应进一步研究,以便采取有效的防控措施,保护人类及畜群的健康。     

Development and Application of a GeXP-multiplex PCR Assay for Detection of Seven Foodborne Pathogenic Bacterias

This experiment was developed to simultaneously detect the seven common foodborne pathogenic bacterias include E. coli O157:H7, Salmonella, V. cholera, L. monocytogenes, C. jejuni, V. Parahemolyticus and S. aureus. Seven pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database. Single and mixed pathogen DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Control group was set up, recombinant plasmids were constructed, and samples of different DNA concentrations were randomly combined to verify the sensitivity, specificity, accuracy and anti-interference of the established GeXP method. To certify the accuracy and reliability of the GeXP assay, it was evaluated using 120 clinical specimens that were compared with the single PCR. The obtained results showed that the corresponding specific fragments of genes were amplified by the single and the multiplex GeXP PCR assay. The detection limit of GeXP was 10³ copies·μL^-1 when all of seven bacterial pathogens were detected. The results of the interference assay showed the presence of specific amplification peaks when different templates. The detection rate of the GeXP multiplex PCR method was 2.50% (3/120)-15.83% (19/120) while the conventional PCR was 2.50% (3/120)-15.00% (18/120), and GeXP multiple PCR detected 8 more positive cases, which means that, the GeXP was more sensitive and accurate in the detection of the clinical samples. In conclusion, this GeXP-based multiplex PCR is a high-throughput, specific and sensitive test to detect seven common foodborne pathogenic bacterias. This assay provides a method in rapid molecular diagnosis for mix clinical seven common foodborne pathogenic bacterias.     

A Droplet Digital PCR Method for Detection of Newcastle Disease Virus

This experiment was conducted to establish a droplet digital PCR (ddPCR) method for the detection of Newcastle disease virus (NDV). A ddPCR method was developed, which the primers and probes were designed based on the conservative regions of F gene of NDV. The concentration of primer and probe, the annealing temperature in ddPCR reaction were optimized. The sensitivity, specificity and reproducibility of ddPCR method were evaluated. In results, the optimal primer concentration and the probe concentration were 900 and 250 nmol·L^-1, the optimum annealing temperature was 55℃. The detection limit of ddPCR method was 1.8 copies·μL^-1 with a good linear response, it had no cross reaction with other six viruses (include IBV), the coefficient of variation of sample repetition was 2.4%. All the results showed that NDV ddPCR was sensitive and specific, it was suitable for quantitative detection of clinical samples infected with NDV.     

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。