High-throughput detection of West Nile virus RNA

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.

FIG. 1

Quantitation of WNV by real-time RT-PCR assay. C_T values are plotted versus the log of a known amount of WNV (in PFU) (A) or in vitro transcribed RNA (B).

FIG. 2

Standard RT-PCR and nested PCR for WNV RNA detection. (A) Comparison of sensitivities of RT-PCR and the nested PCR assay. A titrated amount of WNV (in PFU) was tested by standard RT-PCR (lanes 2 to 8) and nested RT-PCR (lanes 10 to 16). The amounts of viral RNA (in PFU) are indicated above each lane. Lane 8, 80 PFU of viral RNA was subjected to PCR without RT. All RT-PCR mixtures contained Q solution. Lanes 1 and 9, 100-bp marker. (B) Addition of Q solution to the RT-PCR mixture increases the sensitivity by more than 10-fold. In the absence of Q solution, a standard RT-PCR was performed with a titrated amount of virus, as indicated above each lane (lanes 2 to 4). Without Q solution, no product was detected with 0.8 PFU of virus (Lane 3). Lane 1, 100-bp marker. (C) Standard RT-PCR analysis of specimens. Lanes 2 and 3, mosquito specimens; lane 4, a bird specimen; lane 1, 100-bp marker.

FIG. 3

ABI Prism 6700 workstation extracts RNA as efficiently as the RNeasy method. Uninfected bird tissues spiked with titrated amounts of WNV were extracted by RNeasy methods (left panels) or with the ABI Prism 6700 workstation (right panels). The recovered RNA was subjected to standard RT-PCR amplification and analyzed on agarose gels stained with ethidium bromide. The amounts of WNV spiked into the sample (in PFU) are indicated above each lane. (A and B) Samples extracted from kidney and heart tissues, respectively. Lanes 1, 100-bp markers.