High-throughput detection of West Nile virus RNA

Pei-Yong Shi, E. B. Kauffman, Ping Ren, A. Felton, J. H. Tai, A. P. Dupuis, S. A. Jones, K. A. Ngo, D. C. Nicholas, J. Maffei, G. D. Ebel, K. A. Bernard, L. D. Kramer

Research output: Contribution to journalArticle

170 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1264-1271
Number of pages8
JournalJournal of Clinical Microbiology
Volume39
Issue number4
DOIs
StatePublished - 2001
Externally publishedYes

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West Nile virus
Reverse Transcription
RNA
Polymerase Chain Reaction
New England
Viral RNA
Robotics
Nucleic Acids
Disease Outbreaks
Limit of Detection

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Shi, P-Y., Kauffman, E. B., Ren, P., Felton, A., Tai, J. H., Dupuis, A. P., ... Kramer, L. D. (2001). High-throughput detection of West Nile virus RNA. Journal of Clinical Microbiology, 39(4), 1264-1271. https://doi.org/10.1128/JCM.39.4.1264-1271.2001

High-throughput detection of West Nile virus RNA. / Shi, Pei-Yong; Kauffman, E. B.; Ren, Ping; Felton, A.; Tai, J. H.; Dupuis, A. P.; Jones, S. A.; Ngo, K. A.; Nicholas, D. C.; Maffei, J.; Ebel, G. D.; Bernard, K. A.; Kramer, L. D.

In: Journal of Clinical Microbiology, Vol. 39, No. 4, 2001, p. 1264-1271.

Research output: Contribution to journalArticle

Shi, P-Y, Kauffman, EB, Ren, P, Felton, A, Tai, JH, Dupuis, AP, Jones, SA, Ngo, KA, Nicholas, DC, Maffei, J, Ebel, GD, Bernard, KA & Kramer, LD 2001, 'High-throughput detection of West Nile virus RNA', Journal of Clinical Microbiology, vol. 39, no. 4, pp. 1264-1271. https://doi.org/10.1128/JCM.39.4.1264-1271.2001
Shi, Pei-Yong ; Kauffman, E. B. ; Ren, Ping ; Felton, A. ; Tai, J. H. ; Dupuis, A. P. ; Jones, S. A. ; Ngo, K. A. ; Nicholas, D. C. ; Maffei, J. ; Ebel, G. D. ; Bernard, K. A. ; Kramer, L. D. / High-throughput detection of West Nile virus RNA. In: Journal of Clinical Microbiology. 2001 ; Vol. 39, No. 4. pp. 1264-1271.
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