Development Of FAFLP For Genotyping Of Yersinia Pestis

Aim:Amplified fragment length polymorphisms(AFLPs) is polymerase chain reaction(PCR)-based marker with high-resolution for the rapid screening of genetic diversity. It is a new DNA fingerprint technology. AFLP technology by using primers labeled with fluorescent dye is FAFLP. It was used to analyze 261 strains of yersinia pestis from 11 natural foci, purpose to know the relativity of AFLP genotype and epidemiology and natural circumstance, detection any emerging strains that might be introduced accidentally from another country, or by bioterrorism. Method:261 strains of Y. pestis were selected from 10 natural foci isolated at different time from different hosts. The chromosomal DNA were purified by using conventional phenol-chloroform extraction and the concentration were determined by UV spectrometer. In addition, two isolates were subcultured three times and at three different time, DNA were extracted as above.Genomic DNA of two strains were digested with Apa I /Taq I, EcoR I /Mse I and HindIII/Taq I restriction endonuclease respectively. The optimal endonuclease combination were selected.Five primers of EcoR I , nine primers of Mse I and relative adapters were designed based on Janssen et al. Genomic DNA were digested with rare-cutter restriction endonuclease EcoR I and frequent-cutter restriction endonuclease Mse I and subsequent ligation with specific adapters to all restriction fragments. The restriction-ligation fragments were diluted with TE, mixture can be used as the template for selective AFLP reaction. The selective amplicon was added deionied formamide and GeneScan-500(ROX) size standard denaturation and electrophoresis in ABI PRISM 3100 Genetic Analyzer, different electrophoresis parameter and analysis module were selected to analyze the data, data was collected with Datacollection software and analyzed with GeneScan Software, RAPD, PHYLIP and Treeview software.45 pairs primers were tested to select the optimal. In order to determine optimal PCR conditions, The concentration of Mg2+, template, dNTPs, primer and Taq polymerase were optimized. Result:The results of electrephoresis EcoR I /Mse I combination was optimal.The results of capillary electrophoresis for products of PCR with 45 pairs of primers EcoR. I -0/ Mse I -A,C,G,T,CA was optimal and EcoR I -0/ Mse I -C was selected for further tests.The optimization results of PCR indicated that the concentration of Mg2+ affected strongly on the result, too low concentration may led to a few bands and too high led to Pull-up peak and the reduces signal-noise ratio, Mg2+ concentration with 2mmol/L and 2.5mmol/L was optimal. In this study 2.5mmol/L was selected. The concentration of template affect the results also, too low concentration led to a few bands and weak singal, too high led to strong singal over the range of test and signal: noise reduced. 10ng were selected in this study. The results were also affected by the concentration of dNTPs, primer and Taq polymerase.261 strains from 10 natural foci in china were analyzed based on optimized parameters, the results indicate that Yersinia pestis is a highly homologous group with similarity over 97%. 261 strains were grouped into 7 AFLP genotypes, distributing unevenly in 10 natural foci .however, there is one or several predominant genotypes in each focus. AFLP genotypes are related with natural foci and epidemiology.The AFLP results of three subcultures from two strains indicate different electropherogram and group into different group, which suggested that Yersinia pestis is a group with high gene fluidity. Conclusion:FAFLP isn't suitable for genotyping Y. pestis due to the genomic fluidity of thisbacterium, although FAFLP technology per se is a perfect fingerprinting technology.Other new menthod should be explored.