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Journal of Clinical Microbiology, August 2009, p. 2363-2368, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.r00092-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR {triangledown}

Sujit R. Jangam, Douglas H. Yamada, Sally M. McFall, and David M. Kelso*

Center for Innovation in Global Health Technologies, Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208

Received 15 January 2009/ Returned for modification 9 March 2009/ Accepted 28 May 2009

PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 µl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.

* Corresponding author. Mailing address: Center for Innovation in Global Health Technologies, Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208. Phone: (847) 467-2167. Fax: (847) 491-4928. E-mail: kels{at}northwestern.edu

{triangledown} Published ahead of print on 3 June 2009.

Journal of Clinical Microbiology, August 2009, p. 2363-2368, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.r00092-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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