肝螺杆菌多重PCR检测方法的建立及应用

Development and application of multiplex PCR assay for detection of Helicobacter hepaticus

目的建立肝螺杆菌的多重PCR检测方法,对中国动物肝螺杆菌进行检测。方法以毒力基因flaB基因、ureA基因和cdtB基因和cdtC基因作为靶基因,建立检测肝螺杆菌的多重PCR方法。对肝螺杆菌、空肠弯曲菌、幽门螺杆菌、沙门氏菌、志贺氏菌、小肠结肠炎耶尔森菌、大肠埃希氏菌和铜绿假单胞杆菌抽提的DNA进行多重PCR扩增。应用本研究建立的多重PCR检测方法对482只动物(其中:海南30只猴、江苏34只小鼠和北京32只猴、30头猪、66只犬、13只兔、29只豚鼠、69只大鼠、213只小鼠)进行检测,对扩增出的阳性结果进行测序。同时,对所有样本采用选择性培养基进行肝螺杆菌培养以作对照。结果肝螺杆菌能扩增出各自的特异性条带,而其他参考菌株均未扩增出条带,这表明该方法具有较强的特异性。482份样本中检出43份肝螺杆菌阳性样本,阳性率8.92%(43/482),其中:2只犬(3.03%,2/66)、1只兔(7.69%,1/13)、5只大鼠(7.25%,5/69)和35只小鼠(16.43%,35/247)均扩增出肝螺杆菌毒力基因片段。结果显示,肝螺杆菌flaB基因、ureA基因和cdtB基因和cdtC基因序列与GenBank中的肝螺杆菌ATCC51449的相应基因序列核苷酸同源性高达99%。用选择性培养基能培养出肝螺杆菌。结论中国动物中的犬、兔、大鼠和小鼠均能检出肝螺杆菌flaB基因、ureA基因、cdtB基因和cdtC基因。建立的多重PCR检测方法可作为肝螺杆菌大规模检测的新技术,这为在中国开展肝螺杆菌流行病学调查提供了有效科学工具。

To establish a method of detecting Helicobacter hepaticus in Chinese animals by multiplex PCR. a multiplex PCR assay targeting fla B gene, ure A gene, cdt B gene and cdt gene of Helicobacter hepaticus was developed. DNA were extracted from Helicobacter hepaticus, Helicobacter pylori, Campylobacter jejuni , Salmonella spp, Shigella sp, Yersinia enterocolitica, Escherichia coli, and Pseudornonoas aeruginosa and amplified by multiplex PCR. The prevalence of Helicobacter hepaticus in Chinese animals （30 monkeys from Hainan, 34 mice from Jiangsu and 32 monkeys, 30 pigs, 66 dogs, 3 rabbits, 4 guinee pigs, 25 rats and 69 mice from Beijing） was investingated with the multiplex PCR developed in this study. Meanuhile bacterial selective culture was used as a control for the detection of Helicobacter hepaticus in all samples. The experimental results showed that the species-specific products couldy detected after amplification of the DNA template of Helicobacter hepaticus, in contrast, other bacteria strains could not be detected, suggesting the specificity of the multiplex PCR specific. 516 animal samples were detected by multiplex PCR. The fla B gene, ure A gene, cdt B gene and cdt gene of Helicobacter hepaticus were amplified in 2 of 66 dog specimens （3.03 %, 2/66）, 1 of 13 rabbit specimens （7.69 %, 1/13）, 5 of rat specimens （7.25 ,5/ 69）, and 35 mice specimens（16.43 %, 35/247）. The results showed that the average prevalence of Helicobacterhepaticus in Chinese animals was 8.92 % （43/482）, and the prevalence of mice （16.43 % ） was the highest in all kinds of specimens. The results of sequencing indicated that nucleotide homology of H. hepaticus from Chinese animals with H. hepaticus ATCC 51449 in GenBank was 99 %. Helicobacterhepaticus could be cultivated by Selective medium. It is evident that the fla B gene, ure A gene, cdt B gene and cdt gene of Helicobacterhepaticus can be detected in dog, rabbit rat and mice in China. The multiplex PCR established in this study can be used as a new method for throught-out detection Helicobacter hepaticus , and which may provide an useful and scientific detecting tool to carry out Helicobacter hepaticus epidemiological investigation in China.