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Journal of Clinical Microbiology, April 2001, p. 1264-1271, Vol. 39, No. 4
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.4.1264-1271.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

High-Throughput Detection of West Nile Virus RNA

Pei-Yong Shi,1,* Elizabeth B. Kauffman,1 Ping Ren,1 Andy Felton,2 Jennifer H. Tai,3 Alan P. Dupuis II,1 Susan A. Jones,1 Kiet A. Ngo,1 David C. Nicholas,1 Joseph Maffei,1 Gregory D. Ebel,1 Kristen A. Bernard,1 and Laura D. Kramer1

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 122011; Applied Biosystems, Sample Preparation Systems, Foster City, California 944042; and Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303333

Received 12 December 2000/Accepted 18 January 2001

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

* Corresponding author. Mailing address: Wadsworth Center for Laboratories and Research, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12201. Phone: (518) 473-7487. Fax: (518) 473-1326. E-mail: ship{at}wadsworth.org.

Journal of Clinical Microbiology, April 2001, p. 1264-1271, Vol. 39, No. 4
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.4.1264-1271.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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