Dataset Information

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.

ABSTRACT: Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. The analytical sensitivity of the assay was demonstrated to be 50 colony-forming units/ml for Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (surrogate for Yersinia pestis). The absence of any false-positives demonstrated high analytical specificity. Screening B. anthracis-infected mouse blood samples and uninfected controls demonstrated effectiveness and specificity in a preclinical application. This study represents proof of the concept of microarray technology to screen simultaneously for multiple bioterror pathogens in blood samples.

SUBMITTER: Tomioka K

PROVIDER: S-EPMC1888491 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.

Tomioka Keiko K Peredelchuk Michael M Zhu Xiangyang X Arena Roberto R Volokhov Dmitri D Selvapandiyan Angamuthu A Stabler Katie K Mellquist-Riemenschneider Jenny J Chizhikov Vladimir V Kaplan Gerardo G Nakhasi Hira H Duncan Robert R

The Journal of molecular diagnostics : JMD 20051001 4

Heightened concern about the dangers of bioterrorism requires that measures be developed to ensure the safety of the blood supply. Multiplex detection of such agents using a blood-screening DNA microarray is a sensitive and specific method to screen simultaneously for a number of suspected agents. We have developed and optimized a multiplex polymerase chain reaction microarray assay to screen blood for three potential bioterror bacterial pathogens and a human ribosomal RNA gene internal control. ...[more]

PMID: 16237218

Similar Datasets

Project description:BackgroundBlood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics.ObjectiveThe aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation.MethodsThe study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: coa (staphylocoagulase) in Staphylococcus aureus, cpsA (capsular polysaccharide) in Streptococcus pneumoniae, uidA (beta-D-glucuronidase) in Escherichia coli, oprL (peptidoglycan-associated lipoprotein) in Pseudomonas aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software.ResultsIn phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples.ConclusionsSuccessful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods.International registered report identifier (irrid)PRR1-10.2196/33746.

Dataset Information

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.

Publications

A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets