CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

PeerJ

article

Nuttachat Wisittipanit¹  Chaiwat Pulsrikarn²  Sudarat Srisong³  Rungthiwa Srimora³  Nattinee Kittiwan⁴  Kritchai Poonchareon³ 

[1] Department of Material Engineering, School of Science, Mae Fah Luang University;Department of Medical Sciences, WHO National Salmonella and Shigella Center, National Institute of Health, Ministry of Public Health;Division of Biochemistry, School of Medical Sciences, University of Phayao;Veterinary Research and Development Center

关键词: CRISPR 2 PCR;    HRM typing;    Salmonella typing;    4;    [5];    12:i- subtyping;    Epidemiological research;   

DOI  :  10.7717/peerj.9113

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundNontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans.MethodA small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis.ResultsCRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles.ConclusionCRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

【 授权许可】

CC BY