嗜血杆菌属CODEHOP PCR检测方法的建立及在树鼩检测中的应用  

Establishment and application of CODEHOP PCR assay for detection of Haemophilus spp.in tree shrews

作　　者：邢进[1] 冯育芳[1] 岳秉飞[1] 贺争鸣[1] 孙晓梅[2] 代解杰[2] 

机构地区：[1]中国食品药品检定研究院实验动物资源研究所,北京100050 [2]中国医学科学院/北京协和医学院医学生物学研究所,昆明650118

出　　处：《中国人兽共患病学报》2015年第11期994-1000,共7页Chinese Journal of Zoonoses

基　　金：国家科技支撑计划项目(No.2014BAI01B01)资助~~

摘　　要：目的建立树鼩中嗜血杆菌的CODEHOP PCR快速检测方法,为树鼩微生物质量控制提供参考。方法应用CODEHOP在线简并引物设计工具,比对NCBI中6株嗜血杆菌的外膜蛋白序列设计简并引物。通过4株嗜血杆菌标准菌株和24株它属菌株进行特异性和敏感性评价,将建立CODEHOP PCR检测方法,应用于树鼩嗜血杆菌的检测。结果简并引物HF1/HR8和HF3/HR3扩增标准菌株副鸡嗜血杆菌(ATCC 29545)、溶血嗜血杆菌(ATCC 33390)、流感嗜血杆菌(ATCC33391)、副流感嗜血杆菌(ATCC 33392)的目的片段分别为506bp^535bp和344bp^390bp,经测序和NCBI Blast比对,结果准确。两对引物组合,能够有效的区分嗜血杆菌属菌株与它属菌株,检测限最低可达2.1pg/μL。在受试的野生树鼩呼吸道样本中嗜血杆菌阳性率为33.3%(20/60)。结论所建立的方法具有较好的特异性和敏感性,操作简便,能够用于动物样品的嗜血杆菌检测。We established a rapid detection method of Haemophilus spp. in tree shrews, and provided a reference for tree shrews microbiological quality control. According to the outer membrane protein （OMP） multiple alignments of 6 different Haemophilus spp. published in NCBI, a few pairs of degenerate primers were designed by CODEHOP designer online. Then, the specificity and sensitivity of designed CODEHOPs were evaluated by 4 standard Haernophilus strains and 24 other genus strains. And Haernophilus spp. in the tree shrews was detected by established CODEHOP PCR method. Results showed that degenerate primers HF-1/HR-8 and HF-3/HR3 could produce target fragments of 506 - 535 bp and 344 - 390 bp by amplifying H. paragallinarum （ATCC29545）, H. haemol yticus （ATCC33390）, H. in fluenzae （ATCC33391）, H. parainfluenzae （ATCC33392）, respectively. Amplified nucleotides were sequenced and Blast in NCBI, which proved that results were accu- rate. It can effectively distinguish Haemophilus spp. and other genus together with two pairs of primers. The lowest detection limit of this method was 2.1 pg/uL. The 60 respiratory specimens of wild tree shrews were tested for Haemophilus spp. that positive rate was 20/60. In conclusion, the CODEHOP PCR performs well for detection Haernophilus spp. in animal clinical specimens. It is easy to operate, with good specificity and sensitivity, and has practical application value.

关 键 词：嗜血杆菌 CODEHOP PCR 树鼩 简并引物 检测 

分 类 号：R378[医药卫生—病原生物学]