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Development of a PCR-enzyme immunoassay oligoprobe detection method for Toxoplasma gondii oocysts, incorporating PCR controls.

Authors
Schwab-KJ; McDevitt-JJ
Source
Appl Environ Microbiol 2003 Oct; 69(10):5819-5825
NIOSHTIC No.
20037331
Abstract
Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T. gondii infection. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of T. gondii nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters.
Keywords
Infection-control; Exposure-assessment; Humans; Bacteria; Bacterial-disease; Bacterial-infections; Water-analysis; Water-sampling; Immunological-tests; Immunology; Immunotoxins; Laboratory-testing
Contact
Kellogg J. Schwab, Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, 615 N. Wolfe St., Room W6001, Baltimore, MD 21205
CODEN
AEMIDF
Publication Date
20031001
Document Type
Journal Article
Email Address
kschwab@jhsph.edu
Funding Type
Grant
Fiscal Year
2004
NTIS Accession No.
NTIS Price
Identifying No.
Grant-Number-T42-OH-008428
Issue of Publication
10
ISSN
0099-2240
Source Name
Applied and Environmental Microbiology
State
MD
Performing Organization
Johns Hopkins University
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