title: Development of a candidate method for forensic microbial genotyping using multiplex pyrosequencing combined with a universal biotinylated primer.
abstract: Bacterial genotyping can be used for crime scene investigations and contribute to the attribution of biological attacks for microbial forensics. PyroMark ID Pyrosequencer as an accurate detection platform for single nucleotide polymorphisms (SNPs) has been applied to identify and resolve microorganisms involved in closely Escherichia coli O157:H7 (E. coli O157:H7). To explore more applications and improve the efficiency for pyrosequencing in this field, we developed a method integrated multiplex pyrosequencing with a universal primer. Two multiplex pyrosequencing assays with a universal biotinylated primer were designed to analyze five SNPs located in four gene of E. coli O157:H7 strain. The accuracy of the established assays was validated by genotyping reference strain E. coli O157:H7 EDL933 and E. coli K-12. We also demonstrated that two multiplex pyrosequencing assays were specific and sensitive for genotyping closely related E. coli O157 strains. Reproducibility of results and multiplexing capability were evaluated by a comparison of this method with the monoplex pyrosequencing. Furthermore, these two multiplex pyrosequencing assays have been successfully applied to detect 11 E. coli O157 strains isolated from 1504 Chinese livestock samples. This method reduces costs and time consumption in the process of pyrosequencing analysis, and potentially serves as a rapid tool and reliable candidate strategy for the microbial identification and other genotyping application.
authors.affiliation: Institute of Forensic Medicine, West China College of Preclinical and Forensic Medicine, Sichuan University (West China University of Medical Sciences), PO Box 610041, No. 16, Section 3, RenMin Nan Road, Chengdu, Sichuan, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Department of Clinical Microbiology and Immunology, Faculty of Medical Laboratory Science, Third Military Medical University, PO Box 400038, No. 30, Gaotanyan Street, Shapingba District, Chongqing, PR China.
authors.forename: Xuhu
authors.initials: X
authors.lastname: Mao
authors.affiliation: Department of Forensic Science, School of Basic Medical Sciences, Central South University, PO Box 410000, No. 87, XiangYa Road, Changsha, Hunan, PR China.
authors.forename: Lagabaiyila
authors.initials: L
authors.lastname: Zha
authors.affiliation: Institute of Forensic Medicine, West China College of Preclinical and Forensic Medicine, Sichuan University (West China University of Medical Sciences), PO Box 610041, No. 16, Section 3, RenMin Nan Road, Chengdu, Sichuan, PR China.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Institute of Forensic Medicine, West China College of Preclinical and Forensic Medicine, Sichuan University (West China University of Medical Sciences), PO Box 610041, No. 16, Section 3, RenMin Nan Road, Chengdu, Sichuan, PR China. Electronic address: yunlibing@scu.edu.cn.
authors.forename: Libing
authors.initials: L
authors.lastname: Yun
mesh_terms: D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D053803:Forensic Genetics; D005838:Genotype; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Escherichia coli O157:H7; Microbial forensics; Pyrosequencing; Single nucleotide polymorphism; Universal primers