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| 贵州省畜禽源葡萄球菌和链球菌快速鉴定双重PCR方法的建立及应用 | |
| 引用本文： | 张云丹,马光强,岳筠,周碧君,文明,程振涛.贵州省畜禽源葡萄球菌和链球菌快速鉴定双重PCR方法的建立及应用[J].中国畜牧兽医,2019,46(3):873-880. |
| 作者姓名： | 张云丹 马光强 岳筠 周碧君 文明 程振涛 |
| 作者单位： | 1. 贵州大学动物科学学院, 贵阳 550025; 2. 贵州动物疫病与兽医公共卫生重点实验室, 贵阳 550025; 3. 贵州省动物疫病预防控制中心, 贵阳 550008 |
| 基金项目： | 贵州省农业攻关项目（黔科合NY[2014]3042号）；贵州省研究生教育创新计划项目（GZZ2017002）；贵州省科技创新人才团队项目（黔科合人才团队[2015]4016号） |
| 摘 要： | 为建立一种可准确、快速鉴定畜禽临床病例常见病原葡萄球菌和链球菌的双重PCR方法,本试验选取葡萄球菌的nuc基因和链球菌的EF-TU基因保守片段分别设计合成了1对特异性引物,构建可同时快速鉴别葡萄球菌和链球菌的双重PCR体系,并进行反应条件优化,筛选出最佳引物浓度和退火温度;应用该方法对其他73株革兰氏阳性临床分离细菌进行检测,评价该方法的特异性;将培养的葡萄球菌和链球菌倍比稀释计数后鉴定检测方法的敏感性;应用该检测方法对贵州省部分畜禽葡萄球菌和链球菌临床分离样本进行检测。结果显示,所建立的方法最佳引物添加量均为1μL,最佳退火温度为56℃;其他73株供试菌株检测结果均无扩增条带出现,所建双重PCR方法具有较好的特异性;敏感性试验结果显示,葡萄球菌和链球菌敏感度分别达1.50 ng/μL和1.44 pg/μL;临床样本复检结果显示,73株临床分离细菌中葡萄球菌40株(54.79%)、链球菌33株(45.21%),与传统细菌分离鉴定方法的符合率为97.26%。本试验建立了一种特异、敏感和快速鉴定贵州省畜禽葡萄球菌和链球菌病原的双重PCR方法,为临床病例快速诊断及流行病学调查提供了有效技术。 |
| 关 键 词： | 葡萄球菌 链球菌 双重PCR |
| 收稿时间： | 2018-07-11 |
Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province |
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| ZHANG Yundan,MA Guangqiang,YUE Jun,ZHOU Bijun,WEN Ming,CHENG Zhentao.Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province[J].China Animal Husbandry & Veterinary Medicine,2019,46(3):873-880. | |
| Authors: | ZHANG Yundan MA Guangqiang YUE Jun ZHOU Bijun WEN Ming CHENG Zhentao |
| Institution: | 1. College of Animal Science, Guizhou University, Guiyang 550025, China; 2. Key Laboratory of Animal Health and Veterinary Public Health, Guizhou Province, Guiyang 550025, China; 3. Guizhou Animal Disease Prevention and Control Center, Guiyang 550008, China |
| Abstract: | In order to establish a duplex PCR method for accurately and quickly identifying common pathogens Staphylococcus and Streptococcus in clinical cases of livestock and poultry,this experiment selected nuc gene of Staphylococcus and EF-TU gene conserved fragments of Streptococcus to design a pair of specific primers,construct a duplex PCR system which could simultaneously identify Staphylococcus and Streptococcus,optimize the reaction conditions,screen out the optimal primer concentration and annealing temperature;Using this method to detect 73 other gram-positive clinical isolates,the specificity of the method was evaluated;The sensitivity of the detection method was determined by diluting and counting the cultured Staphylococcus and Streptococcus;The detection method was used to detect the clinical isolates of Staphylococcus and Streptococcus of livestock and poultry in Guizhou province.The results showed that the optimal primer addition amount was 1 μL,and the optimal annealing temperature was 56 ℃.The other 73 strains showed no amplification bands,and the duplex PCR method was better.The specificity of the sensitivity test showed that the sensitivity of Staphylococcus and Streptococcus were 1.50 ng/μL and 1.44 pg/μL,respectively.The retest results of clinical samples showed that there were 40 strains of Staphylococcus (54.79%) and 33 strains of Streptococcus (45.21%) in 73 clinical isolates,the coincidence rate with traditional bacteria separation and identification method was 97.26%.This experiment established a duplex PCR method for specific,sensitive and rapid identification of Staphylococcus and Streptococcus pathogens in Guizhou province,providing effective techniques for rapid diagnosis and epidemiological investigation of clinical cases. |
| Keywords: | Staphylococcus Streptococcus duplex PCR |
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