The Construction Of The Database And Mining System Of Specific Markers In Foodborne Pathogens

The surveillance of foodborne pathogens has shown the urgent need for rapid and dependable methods to detect and characterize the organisms in food and clinical environments. Accurate and timely detection and identification of foodborne pathogens is paramount for increasing protection level of public health, and thus any screening method must be sensitive and specific, as well as rapid. Conventional microbiological quantification techniques such as the plate count methods and biochemical identification methods are time-consuming and they generally require 5 to 6 days to finish the whole process. In recent years, various nucleic acid-based molecular methods, such as Polymerase Chain Reaction (PCR) method and DNA-based microarray method, have been developed for the rapid detection and identification of foodborne pathogens because of simplicity in operation, stable detection results, and savings in time. Unfortunately, some obstacles such as target sequence variation and horizontal gene transfer result in false positive and/or negative misidentification during the detection and characterization of foodborne pathogens. Suitable molecular detection markers are still not abundant enough for the development of such a high-throughout method. Therefore, the power of nucleic acid-based molecular methods has been limited in the specificity and number of detection markers.In this study, aimed on presently existing problems about nucleic acid-based molecular methods for foodborne pathogens, a series researches were carried out. The research contents and results are listed as follows.1. The development of the tool of SMM-system for mining specific markers of foodborne pathogens.This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of foodborne pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. SMM-system integrates, by default, the BLAST program, with a greatly improved efficiency over conventional BLAST software. The design work of SMM-system is performed in a pipelined fashion, where four steps were sequentially executed for mining specific markers and homologous sequences. SMM-system is a high-throughput specific markers generation tool which can generate genus-, species-, serogroup- and even serovar-specific DNA sequences as candidate detection markers. It will be applied in nucleic acid-based molecular methods for detection and identification of foodborne pathogens. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html.2. Mining of species-specific detection targets in the foodborne pathogen Vibrio parahaemolyticus by comparative genomic analysis and the irgB gene was selected for the development of a multiplex PCR assay.23 protein-coding sequences were identified by the SMM-system as V. parahaemolyticus-specific candidate markers. We targeted the irgB gene (vp2603), coding for iron-regulated virulence regulatory protein IrgB, in order to develop a PCR method for the detection of V. parahaemolyticus. PCR specificity was determined by amplification of 293 V. parahaemolyticus templates and by the loss of a PCR product with 11 strains from other Vibrio species and 35 non-Vibrio bacterial strains. The PCR assay had the 369-bp fragment and the sensitivity of 0.17 pg purified genomic DNA from V. parahaemolyticus. Furthermore, a multiplex PCR assay targeting irgB, tdh and trh genes was developed for the detection of total and virulent strains of V. parahaemolyticus. These data indicated that the irgB gene is a new and effective marker for the detection of V. parahaemolyticus. In addition, this study demonstrates that SMM-system has a powerful application in identifying specific markers for the detection and identification of bacterial pathogens.3. The identification and PCR evaluation of Salmonella enterica-specific makers.The utility of SMM-system was confirmed by its application to identify 214 S. enterica-specific protein-coding sequences (CDSs). Among these S. enterica specific CDSs, 59 were designated hypothetical proteins, 64 were identified as putative genes, and 91 have been characterized for their functions. Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Seven specific primer pairs targeting respectively SC1286, SC1431, SC1598, SC2172, SC2225, SC2471 and SC4361 were used for the PCR assay in artificially contaminated milk samples. After the enrichment for 10 h, all PCR assays yielded positive results in artificially contaminated milks. And the sensitivity of these PCR assays was less than 1 cfu/ml. Three specific primer pairs were chosen to develop a multiplex PCR assay. These validated primer sets will be useful for surveillance of S. enterica infections in diagnostic laboratories. The identification of these genes in S. enterica will not only provide candidate specific targets for diagnostic application but also benefit for the understanding of the metabolic behavior unique to S. enterica. 4. The construction of the SMDB database focused on specific markers of foodborne pathogen.The 1052 bacterial genomes (all.fna.tar.gz file), their CDSs (all.ffn.tar.gz file) and CDS annotation data (all.ptt.tar.gz file) used in this study were downloaded from the NCBI bacterial genome resource on March 1, 2010 (ftp://ftp.ncbi.nih.gov/genomes/bacteria/). The genomic datas from 150 strains in 23 foodborne pathogenic bacteria species were extracted from GenBank. We used the free tool of SMM-system to identify specific markers of 23 foodborne pathogenic bacteria species and built up the SMDB database. The SMDB database primarily consists of three main contents: 16 foodborne illness, 23 foodborne pathogens and 6588 specific markers and 852 detection related references. SMDB is a free web-accessible database that enables researchers to quickly access specific markers of foodborne pathogen and related information. This database is very useful in discovering information necessary for diagnosis, detection and treatment of various foodborne illness, preventing foodborne illness and outbreaks. The SMDB database is available at http://202.120.41.154/.