High-throughput detection of west Nile virus RNA

Shi, PY Kauffman, EB Ren, P Felton, A Tai, JH Dupuis, AP Jones, SA Ngo, KA Nicholas, DC Maffei, J Ebel, GD Bernard, KA Kramer, LD

Py. Shi et al., High-throughput detection of west Nile virus RNA, J CLIN MICR, 39(4), 2001, pp. 1264-1271

24

INGLESE

Article

Clinical Immunolgy & Infectious Disease",Microbiology

JOURNAL OF CLINICAL MICROBIOLOGY

0095-1137 → ACNP

39

4

2001

1264 - 1271

ISI

0095-1137(200104)39:4<1264:HDOWNV>2.0.ZU;2-1

The recent outbreaks of West Nile virus (WNV) in the northeastern United St ates and other regions of the world have made it essential to develop an ef ficient protocol for surveillance of WNV. In the present report, we describ e a high-throughput procedure that combines automated RNA extraction, ampli fication, and detection of WNV RNA. The procedure analyzed 96 samples in ap proximately 4.5 h, A robotic system, the ABI Prism 6700 Automated Nucleic A cid workstation, extracted RNA and set up reactions for real-time reverse t ranscription (RT)-PCR in a 96-well format. The robot extracted RNA with a r ecovery as efficient as that of a commercial RNA extraction kit, A real-tim e RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro tr anscribed RNA, we estimated the detection limit of the real-time RT-FCR to be approximately 10 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 previo us test results, Using internal primers in a nested RT-PCR, we increased th e 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 surveillanc e dramatically increased the speed and efficiency of sample throughput for diagnosis.