Automatic identification of species-specific repetitive DNA sequences and their utilization for detecting microbial organisms

Motivation: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor. Results: We have developed a novel computational method that identifies species-specific repeats from microbial organisms and automatically designs species-specific PCR primers for these repeats. We tested the methodology on 30 randomly chosen microbial species and we demonstrate that species-specific repeats longer than 300 bp exist in all these genomes. We also used our methodology to design species-specific PCR primers for 86 repeats from five medically relevant microbial species. These PCR primers were tested experimentally. We demonstrate that using species-specific repeats as a PCR template region can increase the sensitivity of PCR in diagnostic tests. Availability and Implementation: A web version of the method called MultiMPrimer3 was implemented and is freely available at http://bioinfo.ut.ee/multimprimer3/. Contact: maido.remm@ut.ee Supplementary information: Supplementary data are available at Bioinformatics online.

[1]  K. Holmes,et al.  DNA hybridization technique for the detection of Neisseria gonorrhoeae in men with urethritis. , 1983, The Journal of infectious diseases.

[2]  R. Limberger,et al.  Development of a Genomics-Based PCR Assay for Detection of Mycoplasma pneumoniae in a Large Outbreak in New York State , 2001, Journal of Clinical Microbiology.

[3]  M. López,et al.  Innovative tools for detection of plant pathogenic viruses and bacteria , 2003, International microbiology : the official journal of the Spanish Society for Microbiology.

[4]  D. Crowley,et al.  Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. , 2005, BioTechniques.

[5]  Michal J. Nagiec,et al.  Molecular genetic anatomy of inter- and intraserotype variation in the human bacterial pathogen group A Streptococcus. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A Danchin,et al.  Analysis of long repeats in bacterial genomes reveals alternative evolutionary mechanisms in Bacillus subtilis and other competent prokaryotes. , 1999, Molecular biology and evolution.

[7]  K. Klimpel,et al.  Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR. , 2003, Journal of virological methods.

[8]  D. S. Leal-Klevezas,et al.  Single-step PCR for detection of Brucella spp. from blood and milk of infected animals , 1995, Journal of clinical microbiology.

[9]  Reidar Andreson,et al.  Predicting failure rate of PCR in large genomes , 2008, Nucleic acids research.

[10]  Rodrigo Gouveia-Oliveira,et al.  Genome update: DNA repeats in bacterial genomes. , 2004, Microbiology.

[11]  M. Loeffelholz,et al.  PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid , 1994, Journal of clinical microbiology.

[12]  D. Raoult,et al.  Comparison of PCR and Serology Assays for Early Diagnosis of Acute Q Fever , 2003, Journal of Clinical Microbiology.

[13]  A. Fluit,et al.  False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy , 2004, European Journal of Clinical Microbiology and Infectious Diseases.

[14]  G Achaz,et al.  Study of intrachromosomal duplications among the eukaryote genomes. , 2001, Molecular biology and evolution.

[15]  Jakob Fredslund,et al.  Primique: automatic design of specific PCR primers for each sequence in a family , 2007, BMC Bioinformatics.

[16]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[17]  C. Ison,et al.  Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica. , 1986, Journal of clinical pathology.

[18]  A. V. D. van den Brule,et al.  Evaluation of Conventional and Real-Time PCR Assays Using Two Targets for Confirmation of Results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae Test for Detection of Neisseria gonorrhoeae in Clinical Samples , 2005, Journal of Clinical Microbiology.

[19]  Paul M. Ruegger,et al.  PRISE (PRImer SElector): software for designing sequence-selective PCR primers. , 2008, Journal of microbiological methods.

[20]  Bruce A. Roe,et al.  Genome Sequence of a Nephritogenic and Highly Transformable M49 Strain of Streptococcus pyogenes , 2008, Journal of bacteriology.

[21]  H. Ellerbrok,et al.  Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii , 2006, BMC Microbiology.

[22]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[23]  Eric Coissac,et al.  Origin and fate of repeats in bacteria , 2002, Nucleic Acids Res..

[24]  D. Cowan,et al.  Review and re-analysis of domain-specific 16S primers. , 2003, Journal of microbiological methods.

[25]  D. Ussery,et al.  The Atlas visualization of genomewide information , 2002 .

[26]  J. Chisholm,et al.  Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction , 2006, Food additives and contaminants.

[27]  Maido Remm,et al.  Enhancements and modifications of primer design program Primer3 , 2007, Bioinform..

[28]  Namshin Kim,et al.  QPRIMER: a quick web-based application for designing conserved PCR primers from multigenome alignments , 2007, Bioinform..

[29]  D. Farrell Evaluation of AMPLICOR Neisseria gonorrhoeae PCR Using cppB Nested PCR and 16S rRNA PCR , 1999, Journal of Clinical Microbiology.

[30]  I. Clark,et al.  Fungal molecular diagnostics: a mini review. , 2004, Journal of applied genetics.

[31]  B. Duim,et al.  Multicenter Validation of the cppB Gene as a PCR Target for Detection of Neisseria gonorrhoeae , 2004, Journal of Clinical Microbiology.

[32]  J. SantaLucia,et al.  The thermodynamics of DNA structural motifs. , 2004, Annual review of biophysics and biomolecular structure.

[33]  E. Rocha,et al.  Associations between inverted repeats and the structural evolution of bacterial genomes. , 2003, Genetics.

[34]  C. Robert,et al.  Use of Genome Selected Repeated Sequences Increases the Sensitivity of PCR Detection of Tropheryma whipplei , 2004, Journal of Clinical Microbiology.

[35]  Jakob Fredslund,et al.  PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs , 2005, Nucleic Acids Res..

[36]  Thomas D. Otto,et al.  BMC Bioinformatics BioMed Central Methodology article ReRep: Computational detection of repetitive sequences in genome survey sequences (GSS) , 2008 .