Establishment Of MLVA Genotyping Method Of Leptospira And Preliminary Application

Leptospirosis is a worldwide-distributed zoonosis,endemic in tropical areas.It is caused by the leptospira interrogans.Leptospirasis had prevailed severely in our country.Since it was ranked among stipulated infectious diseases in 1955,several times large pandemics had happened.Recently as blood disaster is increasing continually,there are still sporadic cases or outbreak at different degrees in local areas in every summer and autumn.Our country has resourceful leptospira serogroups and serovars.18 serogroups 75 serovars have been found in leptospira interrogans. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests.The traditional serogroup typing can not satisfy the investigation and treatment of leptospira emergencies because of its tedious work,time consuming and high subjectivity.Recently,various molecular typing systems have been set for studying the molecular epidemiology of Leptospira,including MLST,AFLP,RFLP and PFGE.All of these methods suffer from significant drawbacks,including low discriminatory power,poor reproducibility,and the high level of technical skill required to perform the method.As an alternative to these methods we have developed a multiple locus variable number of tandem repeats analysis(MLVA) based upon variable-number tandem-repeat(VNTR).It is widely used in the field of molecular investigations into the epidemiology.In this study,we aimed to extend MLVA identification of strains to the genotyping of L.interrogans.We designed primers for seven VNTR loci such as VNTR4,VNTR7,V27,V29,V30,V36,V50.The aim is to study the relationship between the VNTR pattern and the geographic area,to find the dominant strains and the source of infection and route of transmission,to study the genome evolution.The discriminatory power of the primers was evaluated on Reference strains and collection strains.In this study,we have amplificated 213 L.interrogans successfully.In contrast,we have verified that no amplification was obtained with DNAs from the saprophytic species L.biflexa with the seven markers.213 L.interrogans were divided into 8 gene groups 66 genotypes.The most encountered serogroup was icterohaemorrhagiae, striped field mouse were the main reservoirs of leptospirosia.The VNTR patterns were related to the geographic area.The dominant strains are different between various regions.Each gene group had predominant molecular pattern in the different regions.Correlation was observed between VNTR clusters,PFGE clusters and serological typing.All representive strains profiles achieved in my experiment could be used to the field of genotyping and tracing the source of infection.Above all,VNTR clusters is related to PFGE clusters and serological typing.MLVA could detect more mutation information.It is simple PCR-based and needs short experimental period.Its cost is low.A further advantage of the MLVA method over PFGE analysis is that the MLVA method could be applied in a non-culture-based situation,i.e.It could be possible to type an organism directly from a Leptospira PCR-positive DNA extract.The results are convenient for Comparison among different Laboratory.Though MLVA is in the preliminary phase,we belive that the combination of MLVA and PFGE can improve the identification of leptospira,which can be applied to the tracing of source of infection,to find the dominant strains,to study the genome evolution.