肺炎克雷伯菌与链球菌双重PCR检测方法的构建  

Establishment of Duplex PCR Assay for the Detection of Klebsiella pneumoniae and Streptococcus

作　　者：王一霖 王燕 梁纤纤 郭明强 樊晓慧 夏俊 苏战强[1,2] WANG Yilin;WANG Yan;LIANG Qianqian;GUO Mingqiang;FAN Xiaohui;XIA Jun;SU Zhanqiang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830000,China;Xinjiang Key Laboratory of New Drug Research and Development for Herbivores,Urumqi 830000,China;Institute of Veterinary Medicine,Xinjiang Academy of Animal Science/Center for Animal Clinical Medicine,Xinjiang Academy of Animal Science,Urumqi 830000,China;Key Laboratory for Prevention and Control of Herbivorous Animal Diseases,Ministry of Agriculture and Rural Areas,Urumqi 830000,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830000 [2]新疆草食动物新药研究与创制重点实验室,乌鲁木齐830000 [3]新疆畜牧科学院兽医研究所/新疆畜牧科学院动物临床医学研究中心,乌鲁木齐830000 [4]农业农村部草食动物疫病防控重点实验室(省部共建),乌鲁木齐830000

出　　处：《草食家畜》2024年第3期51-59,共9页Grass-Feeding Livestock

基　　金：新疆维吾尔自治区“天山英才”培养计划项目“草食家畜群发病防控技术推广”(2023SNGGNT005);新疆维吾尔自治区重大科技专项“新疆畜禽疫病防控体系质量提升工程”(2022273358)。

摘　　要：【目的】旨在建立一种可同时检测肺炎克雷伯菌和链球菌的双重PCR检测方法。【方法】选取肺炎克雷伯菌KP-Khe基因和链球菌EF-Tu基因保守片段,分别合成2对特异性引物,优化双重PCR反应体系,摸索其特异性、最佳退火温度、引物浓度和细菌核酸浓度,并通过该方法对临床样品进行检测验证。【结果】该双重PCR检测方法可有效扩增出肺炎克雷伯菌和链球菌的489bp和197bp的特异性片段,最适退火温度为58℃、最适引物浓度为12ng/µL、最适模板浓度为3ng/µL。将该方法应用于临床对15份样品进行检测,其中2份样品检测出肺炎克雷伯菌呈阳性,1份样品检测出链球菌呈阳性,与分离鉴定结果一致。【结论】该研究建立的双重PCR技术,可为临床诊断肺炎克雷伯菌、链球菌及混合感染提供了一种便捷高效的检测方法,具有一定的应用价值。【Objective】To establish a duplex PCR assay for the simultaneous detection of Klebsiella pneumoniae and Streptococcus.【Methods】Two pairs of specific primers were synthesised by selecting the conserved fragments of Klebsiella pneumoniae KP-Khe gene and Streptococcus EF-Tu gene respectively,and the dual PCR reaction system was optimized to explore its specificity,optimal annealing temperature,primer concentration and bacterial nucleic acid concentration.The method was used to test and verify the clinical samples.【Results】The duplex PCR assay was able to effectively amplify 489 bp and 197 bp specific fragments of Klebsiella pneumoniae and Streptococcus,with the optimal annealing temperature of 58℃,the optimal primer concentration of 12 ng/µL,and the optimal template concentration of 3 ng/µL.The method was applied to 15 clinical samples detection,of which 2 samples were positive for Klebsiella pneumoniae and 1 sample was positive for Streptococcus,which was consistent with the isolation and identification results.【Conclusion】The duplex PCR technique established in this study can provide a convenient and rapid detection method for clinical diagnosis of Klebsiae pneumoniae,streptococcus and mixed infection,which has certain application value.

关 键 词：肺炎克雷伯菌 链球菌 双重PCR 

分 类 号：S852.61[农业科学—基础兽医学]