类鼻疽伯克霍尔德菌全基因组序列检测的研究 - 第三军医大学学报.doc

特异引物PCR鉴定类鼻疽伯克霍尔德菌方法的建立和优化 杨小敏1*，方 瑶2*，顾 江2，陈海3，王海光2，毛旭虎2（570102 海口，海南医学院附属医院感染病教研室1，；400038 重庆，第三军医大学医学检验系临床微生物及免疫学教研室，国家免疫生物制品工程技术研究中心2； 572000 海南 三亚，海南省三亚市人民医院检验科3） [摘要K96243的16S rRNA编码序列为靶序列，通过Primer Premier 5软件搜索特异的引物，以该菌为模板，优化特异引物PCR的退火温度、退火时间和反应循环数等条件。得到最优PCR反应条件后，将该方法用于检测临床分离的类鼻疽伯克霍尔德菌菌株和常见细菌感染病原体（大肠杆菌、痢疾志贺菌、伤寒沙门菌、幽门螺旋杆菌、金黄色葡萄球菌、甲型链球菌和乙型链球菌）评价该方法的有效性和特异性。 结果 共设计3对引物，分别扩增16S rRNA编码序列283~672、760~1 061、771~1 193片段，优化后PCR反应条件为：94 ℃预变性4 min，94 ℃变性40 s，54.5 ℃退火20 s，72 ℃延伸40 s，24个循环，最后延伸1 min；特异性分析发现此方法能将类鼻疽伯克霍尔德菌与其他常见感染病原菌进行有效的鉴别诊断。结论 成功建立并优化一种快速鉴定类鼻疽伯克霍尔德菌Establishment and Optimization of a method to identify Burkholderia pseudomallei based on primer-special polymerase chain reaction Yang Xiaomin 1*, Fang Yao2*, Gu jiang2, Chen Hai3, Wang Haiguang2, Mao Xuhu2 (1Department of infectious diseases, the Hospital Affiliated with Hainan Medical College, Haikou, 570102；2Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, The Third Military Medical University, Chongqing，400038；3Department of laboratory medicine，People’s Hosptial of Sanya, Sanya 572000，China) [Abstract] Objective To Establish and Optimizate of a method to identify Burkholderia pseudomallei based on primer-special polymerase chain reaction. Methods The whole genome of Burkholderia pseudomallei international standard stain K96243 was used as a template to amplify the 16S rRNA encoding gene by polymerase chain reaction (PCR) using special primers, and the PCR conditions (annealing temperature, annealing time, cycles) were optimizated at the same time. Then this method was use to differentiate the Burkholderia pseudomallei from other infectious bacteria and to identify clinic Burkholderia pseudomallei isolates. Results Three pairs of primers were selected to amplify fragments 283-672, 760-1061 and 771-1193 of 16S rRNA respectively. The optimized PCR condition is denature at 94℃ for 40s, annealing at 54.5℃ for 20s, elongation at 72℃ for 40s, 24 cycles. This meth