Establishment Of Molecular Type Method Based On CRISPR In Escherichia Coli

Escherichia coli can live in the intestine tract as a commensal bacteria,nevertheless,some serotypes of which are pathogenic for human beings.Pathogenic Escherichia coli can cause a variety of diseases,such as mild diarrhea,hemorrhagic colitis,hemolytic uremia and urinary tract infection.CRISPR/Cas system acts as a adaptive immunity system,which was highly polymorphic.The polymorphism of CRISPR is used to type Escherichia coli.Objective1 To analyse the characteristics and structure of CRISPR using bioinformatic method,to describe the location relationship between CRISPR/Cas system and IS and to investigate the association between CRISPR1 and CRISPR2.2.2 To construct molecular typing method based on CRISPR in Escherichia coli,to compare the discrimination power among CCT,CLPST,MLST,Serotype and to analyze the relationship among them.3 To predict the serotype of Escherichia coli through CRISPR type and to assess the sensitivity and specificity.Method1 The software of CRT and the web sever of CRISPR finder were used to detect and identify basic characteristics of CRISPR and to extract repeats and spacers.The RNA secondary structure of repeats was investigated using RNA fold sever.In order to search the homologous sequence of spacer,CRISPR Target was applied.The phylogenic and evolutionary tree was obtained by Mega 6.0.2 The information about the serotype of Escherichia coli was achieved by the Serotype Finder sever web.Virulence finder sever web was used to acquire the distribution of virulence genes.The results of MLST type was obtained by the MLST Finder sever web.3 The software of Excel was used to mark the spacer as a unique number and to draw the spacer fingerprint map.The R software was applied to obtain the CRISPR type.4 The discrimination power among CCT,CLPST,MLST,Serotype was measured and assessed by the Simpson Index.The Spearman rank correlation test was used to get the correlation coefficient among CCT type,CLPST type,MLST type,Serotype.Result1 Bioinformatic analysis of CRISPR in Escherichia coli: 7 different kinds of CRISPR loci was identified in 283 strains of Escherichia coli with complete genome.The positive rate of CRISPR1,CRISPR2.1,CRISPR2.2,CRISPR2.3,CRISPR3,CRISPR4,CRISPR3-4 was 81.62%,88.69%,88.70%,7.42%,5.65%,5.65%,93.28%,respectively.IS sequence could be inserted into the adjacent location of CRISPR/Cas system,or into the Cas gene,and even between two repeats.2 The construction of molecular typing method based on CRISPR in Escherichia coli:According to the CCT typing method,430 CCT types were obtained.125 CLPST types were achieved by CLPST type method.The Simpson index of CCT type,CLPST type,MLST type,Serotype were 0.8864,0.8760,0.8581,0.8643,respectively.3 The association between the content and order of spacer and the phage of stx : The phage of stx was present in every CRISPR type.There were no differences in the content and order of spacer for bacteria from different sources,time of separation,districts of isolation.4 The application of CRISPR type to predict the serotype of Escherichia coli: The sensitivity and specificity of predicting these serotype,such as O157:H7 or NM,O104:H4,O16:H48,O5:H9,O111:H8 or NM,O121:H19,O103:H2,O45:H2,could reach more than 95%.However,the sensitivity and specificity of predicting the serotype,O26:H11 or NM,was very low.Conclusion1 The CRISPR/Cas system was widely present in Escherichia coli.2 IS sequence existed in the CRISPR/Cas system.3 The discrimination power of CRISPR type for Escherichia coli was excellent.4 CRISPR could be used to predict the serotype of Escherichia coli.