Development Of CRISPR-based Molecular Typing Method Of Salmonella

Salmonella is one of the major foodborne pathogenic bacterium, moreover Salmonella Enteritidis is the most important Salmonella serovar around the world. Molecular typing for Salmonella isolates can reveal their origin, variation and evolution, providing key support data for hazard tracing, risk assessment and hazard control, which can be of great significance for tracking and monitoring pathogen transmission and disease outbreaks.Based on the CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) sequences, 2 novel rapid, effective and simple typing methods for Salmonella were established in this study including CSST(CRISPR initial three Spacers Sequence Typing) and TCSST(Target-CRISPR initial three Spacers Sequence Typing), and further evaluated in the application of molecular typing for Salmonella spp.First of all, CRISPR1 primer pair and CRISPR2 primer pair were designed according to the conserved upstream and downstream sequences of CRISPR. The two CRISPR primer pairs were used to amplify the CRISPR sequences from different Salmonella serovars, and at least 30 kinds of Salmonella serovars could be classified with the CRISPR1 and CRISPR2 spacer sequence profiles. Salmonella-specific target S69 was chosen as the helper sequence to set up TCSST typing method from 8 targets S9, S45, S69, S87, S95, S121, S165 and S200, to improve the discrimination power of CSST typing method when necessary.Four typing methods, traditional CRISPR typing, S69 typing, CSST typing and TCSST typing were used to discriminate a total of 86 Salmonella strains representing 30 serovars, it turned out that the discrimination power of the four methods were 0.9692, 0.8902, 0.9199 and 0.9423, repectively, which demonstrated that four methods were all suitable for typing different Salmonella serovars, esp. traditional CRISPR typing and TCSST. It was notable that CSST typing method was superior to S69 typing method, which can differentiate different serovars(>21 serovars) as well as different isolates from one serovar. Besides, Only three spacers of CRISPR1 / CRISPR2 upstream sequence need to be analysed in CSST, which is easier than traditional CRISPR typing, with sequencing of low cost. Therefore, CSST could be used for the rapid and effective differentiation of different Salmonella serovars.Six typing methods, virulence gene typing, ERIC-PCR typing, CRISPR typing, CLSPT typing, CSST typing and TCSST typing were used to discriminate 90 Salmonella Enteritidis isolates from the year of 2008 to 2012 from different sources in Shanghai. Consequently, each type number was 7, 1, 6, 1, 4 and 12, respectively, and their discrimination power was 0.4092, 0, 0.1293, 0, 0.0871 and 0.7506, accordingly, which proved that TCSST typing was the best method. What’s more, TCSST types were correlated with the sources and isolation years of S. Enteritidis isolates. We also discussed the relationship between TCSST type and virulence genes. In addition, when the number of strains was amplified to 198 Salmonella Enteritidis strains, 31 TCSST types were obtained, and the value of discrimination power was 0.6555; it proved that TCSST had the potential to be applied in the S. Enteritidis typing.All in one, two novel molecular typing methods were developed in this study as CSST and TCSST; CSST typing could be used in the classification of different Salmonella serovars with advantages of simple operation and low cost; and when S69 was involved, TCSST typing could be used for the discrimination of different Salmonella isolates from one serovar such as S. Enteritidis.