Simultaneous pathogen detection and antibiotic resistance characterization using SNP-based multiplexed oligonucleotide ligation-PCR (MOL-PCR).
Extensive use of antibiotics in both public health and animal husbandry has resulted in rapid emergence of antibiotic resistance in almost all human pathogens, including biothreat pathogens. Antibiotic resistance has thus become a major concern for both public health and national security. We developed multiplexed assays for rapid, simultaneous pathogen detection and characterization of ciprofloxacin and doxycycline resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. These assays are SNP-based and use Multiplexed Oligonucleotide Ligation-PCR (MOL-PCR). The MOL-PCR assay chemistry and MOLigo probe design process are presented. A web-based tool - MOLigoDesigner (http://MOLigoDesigner.lanl.gov) was developed to facilitate the probe design. All probes were experimentally validated individually and in multiplexed assays, and minimal sets of multiplexed MOLigo probes were identified for simultaneous pathogen detection and antibiotic resistance characterization.
Song
Jian
J
Biosecurity and Public Health (B-7), Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87544, USA. jian@lanl.gov
Li
Po-E
PE
Gans
Jason
J
Vuyisich
Momchilo
M
Deshpande
Alina
A
Wolinsky
Murray
M
White
P Scott
PS
D000818:Animals; D001408:Bacillus anthracis / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity; D002939:Ciprofloxacin / Q000494:pharmacology; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005604:Francisella tularensis / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity; D006801:Humans; D020407:Internet; D015336:Molecular Probe Techniques; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015010:Yersinia pestis / Q000187:drug effects / Q000235:genetics / Q000472:pathogenicity
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D002939:Ciprofloxacin; D004318:Doxycycline