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Title: Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

Abstract

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt)more » and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

Authors:
; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab., CA (US)
Sponsoring Org.:
USDOE Office of Defense Programs (DP) (US)
OSTI Identifier:
793859
Report Number(s):
UCRL-ID-139001
TRN: US200302%%657
DOE Contract Number:  
W-7405-Eng-48
Resource Type:
Technical Report
Resource Relation:
Other Information: PBD: 5 May 2000
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AMPLIFICATION; BACILLUS; DETECTION; EVALUATION; FLUORESCENCE; IMMUNOASSAY; LATEX; MICROORGANISMS; MICROSPHERES; NUCLEIC ACIDS; SENSITIVITY; TESTING

Citation Formats

Venkateswaran, K S, Nasarabadi, S, and Langlois, R G. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer. United States: N. p., 2000. Web. doi:10.2172/793859.
Venkateswaran, K S, Nasarabadi, S, & Langlois, R G. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer. United States. doi:10.2172/793859.
Venkateswaran, K S, Nasarabadi, S, and Langlois, R G. Fri . "Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer". United States. doi:10.2172/793859. https://www.osti.gov/servlets/purl/793859.
@article{osti_793859,
title = {Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer},
author = {Venkateswaran, K S and Nasarabadi, S and Langlois, R G},
abstractNote = {Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.},
doi = {10.2172/793859},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2000},
month = {5}
}

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