Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish

Abstract

Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "Norwalk-like" viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR-oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

FIG. 1

RT-PCR coamplification of internal standard amplicons with NV RNA using rTth, NV p35 and p36 primers, and UNG. Lanes 1 to 8 contain 100 copies of NV (10 μl of a 10⁻³ dilution of NV RNA) and 10 μl of a 10-fold dilution series of PCR product amplified with dUTP (lane 1, 10⁻² dilution of PCR product [10⁸ PCRU], through lane 7, 10⁻⁸ dilution of PCR product [10² PCRU]). Lane 9, negative reagent control; lane 10, 123-bp molecular weight marker.

FIG. 2

DEIA using probe SR65 (A) and Southern blot using probe NVp69 (B) detection of pooled RT-PCR amplification of NV half-log₁₀ dilution series end-point determination. RT-PCR conditions included use of rTth, UNG, NV p35 and p36 primers, and addition of 50 copies of internal standard (except lane 7).

FIG. 2

DEIA using probe SR65 (A) and Southern blot using probe NVp69 (B) detection of pooled RT-PCR amplification of NV half-log₁₀ dilution series end-point determination. RT-PCR conditions included use of rTth, UNG, NV p35 and p36 primers, and addition of 50 copies of internal standard (except lane 7).

FIG. 3

SR65 probe DEIA results of RT-PCR amplicons generated by RT-PCR using rTth, UNG, NV p35 and p36 primers, and 50 to 100 copies of internal standard. Viral RNA was recovered from stools and oysters using described methods prior to amplification.

FIG. 4

Southern transfer oligoprobe and DEIA detection following rTth RT-PCR amplification of internal standard and NV bioaccumulated in oysters. RT-PCR of 10-fold (lanes 1, 3, 5, 7, 9, and 11) and 100-fold (lanes 2, 4, 6, 8, 10, and 12) dilutions of RNA of individual oyster (Oyst) extracts obtained following dissection of the digestive diverticula from oysters after 24 h of bioaccumulation with 10⁵ NV PCRU/5 liters. Lanes 1 to 14 contain 100 copies of internal standard. Other lanes include 100 copies of an internal standard (int std) control (lane 13), an NV RT-PCR-positive control (lane 14), a negative reagent control (lane 15), and a digoxigenin-labeled molecular size marker (Marker VIII; Boehringer-Mannheim) (lane 16). Numbers at the right are molecular sizes in base pairs. Below the Southern blot are the DEIA OD readings, with underlined numbers corresponding to positive DEIA samples.