Title: Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

Abstract

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus withmore » 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Authors:

Thissen, James B. ^[1]; McLoughlin, Kevin ^[2]; Gardner, Shea ^[1]; Gu, Pauline ^[1]; Mabery, Shalini ^[1]; Slezak, Tom ^[2]; Jaing, Crystal ^[1]

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+ Show Author Affiliations

1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Physical and Life Sciences Directorate
2. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Computations Directorate

Publication Date:

Sun Jun 01 00:00:00 EDT 2014

Research Org.:

Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1124829

Report Number(s):

LLNL-JRNL-594212
Journal ID: ISSN 0166-0934; PII: S0166093414000433

Grant/Contract Number:  

W-7405-ENG-48

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Virological Methods

Additional Journal Information:

Journal Volume: 201; Journal Issue: C; Journal ID: ISSN 0166-0934

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES