Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology

Abstract

A bead-based liquid hybridization assay, Luminex(®) 100™, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR-amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty-eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.

Surface plasmon resonance imaging of pathogens: the Yersinia pestis paradigm

“…Some new approaches were applied in the detection of bioterrorism agents. These techniques were principally based on amplified DNA-sequence detection including high-throughput RT-PCR-coupled ESI–MS assay and Luminex assay [ 12 , 13 ]. According to the current state of the art, SPRi is less sensitive than PCR-based techniques [ 20 ] and this sensitivity remains to be improved.…”

“…Also, immunochromatography detection of the Y. pestis -specific F1 antigen [ 9 – 11 ] is used for research, as this diagnostic assay is not widely available. Recently, some new approaches such as high-throughput RT-PCR-coupled ESI–MS assay and Luminex were applied to the detection of bioterrorism agents [ 12 , 13 ]. These techniques required principally amplified DNA and apparently remained time-consuming.…”

RNA and DNA Diagnostics

“…In another area, liquid microsphere microarray was used in sensitive detection of anthrax and its relative pathogens. In the report, two genes from nine anthrax core pathogens were selected and analysed in one reaction (Schweighardt et al 2014). Another study utilised a 15-plex PCR, followed by hybridisation onto oligonucleotide probes conjugated onto microspheres, to detect several species of pathogenic bacteria and some of their serotypes (Zhao et al 2013).…”

Nucleic acid detection technologies and marker molecules in bacterial diagnostics

“…Additionally, a fluorochrome coupled to a reporter molecule quantifies the nucleic acid interaction that has occurred at the microsphere surface [49]. Such suspension array technology in combination with target PCR amplification has been used for multiplexed typing of important animal pathogens [50] and is also suggested for the detection of several human pathogens related to biological warfare and terrorism [51].…”