REP-PCR Genotyping Analysis and Application of Chinese Brucella Isolates
|Course||Microbial and Biochemical Pharmacy|
|Keywords||Brucella REP-PCR Epidemiology|
Brucellosis is infectious disease caused by Brucella’s invasion of the body and vulnerable to infection between humans and animals, being popular not only in China but also around the world. According to statistics, total of25provinces have brucellosis distribution from1993to2009, and the incidence of brucellosis increased by130times has seriously affected the development of animal husbandry.More than a century, the disease has been one of the world’s five largest zooepidemicus.Traditional genotyping methods have divided Brucella into nine species and19subtypes, including B.melitensis species1,2,3subtypes, B.abortus species1,2,3,4,5,6,7,9subtypes,B.suis species1,2,3,4,5subtypes,B.canis species, B.neotomae species, B.ovis species B.microti, B.pinnipediae, B.cetacea based on the difference of host preference, as well as culture and biological characteristics.The representative176epidemic strains of Brucella are chosen from China’s26provinces in the study to analyse the information source of strain;The REP-PCR genotyping methods need to fumble conditions and establish REP-PCR typing technique;The established good genotyping method is applied to identify Brucella strains, obtaining amplification profiles;The result of analysing information source of strain combines the result of REP-PCR genotyping to kown bucellosis’s epidemic trends and popular from1950s times to2000s times for providing the basis for for our reasonable control measures.The result of information source of strain is that B.melitensis species3subtype and B.abortus species1subtype were popular in1950s, B.melitensis species2subtype was popular in1960s, B.abortus species6subtype was popular in1970s, B.canis species was popular in1980s, B.melitensis species1subtype was popular in1990s, B.melitensis species3subtype was popular in00s.To fumble this experiment’the main factors affecting the REP-PCR reaction conditions:template, enzymes, primers, and annealing temperature. The template is selected by using the extraction kit and the purity and concentration can meet the requirements of the amplification; rTaq enzyme has advantages for REP amplification but the other two enzymes can’t amplificate all of the strips;The concentration of primers selected10μm;40℃is the most clear amplification bands. REP-PCR typing method is established at last. B.abortus species and B.melitensis species, part of B.suis species1,3subtypes and B.abortus species are respectively close from Brucella strains clustering results, but the relationship between B.abortus species and B.melitensis species is more closer. There is a close relationship between B.canis species and B.suis species as well as B.abortus species.B.melitensis species3subtype was respectively populer in the50’s and00’s. The region of infected B.melitensis species3subtype was mainly the west and north between50’s-90’s, changing to the eastern, northeastern and southern after00’s. The eastern infections came from the north and the northeast and south of infection came from the west from the genetic relationship. B.abortus species1subtype exited two branches:the relationship of Inner Mongolia, Beijing, Tianjin and Henan strains were nearly and Anhui,Xinjiang and Shanghai strains were nearly.Shanxi, Shanghai, Qinghai, Jilin, Henan, Shandong and Xinjiang emerged B.melitensis species2subtype after60years later and the trendence was consistent with B.melitensis species3subtype. B.abortus species6subtype was mainly popular in the1970s,the main scope including Inner Mongolia, Heilongjiang, Ningxia and Tibet and the relationship of Heilongjiang strains farther than other parts region. B.abortus species3subtype strains were clustered into one branch before the1980s and another branch after the1980s. It exited mainly in Ningxia region before1980s and Inner Mongolia and Xinjiang regions after the1980s. B.canis occured mainly in the eastern coastal cities, such as Shanghai, Jiangsu, Guangxi, Shandong and other places. In1990s, B.melitensis species3subtype was most popular in Inner Mongolia.Inner Mongolia, Xinjiang, Sichuan, Ningxia, Henan, Guangxi, Shandong and so on are most serious areas infected Brucella. We should do well in checking infected areas and eliminating the infected anmimals as well as vaccination work. At same time, people should not contact with sick animals and fur. From epidemiology to develop feasible measures is of great significance to the pr evention and control of brucellosis.