Establishment and application of CODEHOP PCR assay for detection of Haemophilus spp. in tree shrews
XING Jin1, FENG Yu-fang1, YUE Bing-fei1, HE Zheng-ming1, SUN Xiao-mei2, DAI Jie-jie2
1.National Institute of Food and Drug Control, Institute of Laboratory Animal Resources, Beijing 100050, China; 2.Institute of Medical Biology, Chinese Academy of Medicine Sciences and Peking Union Medical College, Kunming 650118, China
Abstract：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 Haemophilus strains and 24 other genus strains. And Haemophilus 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. haemolyticus (ATCC33390), H. influenzae (ATCC33391), H. parainfluenzae (ATCC33392), respectively. Amplified nucleotides were sequenced and Blast in NCBI, which proved that results were accurate. 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 Haemophilus spp. in animal clinical specimens. It is easy to operate, with good specificity and sensitivity, and has practical application value.
邢进， 冯育芳， 岳秉飞， 贺争鸣， 孙晓梅， 代解杰. 嗜血杆菌属CODEHOP PCR检测方法的建立及在树鼩检测中的应用[J]. 中国人兽共患病学报, 2015, 31(11): 994-1000.
XING Jin, FENG Yu-fang, YUE Bing-fei, HE Zheng-ming, SUN Xiao-mei, DAI Jie-jie. Establishment and application of CODEHOP PCR assay for detection of Haemophilus spp. in tree shrews. Chinese Journal of Zoonoses, 2015, 31(11): 994-1000.
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2015, Vol. 31 
