Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes
Why the study?
Does the RT-PCR-OP method accurately detect Norwalk virus in stool specimens compared to standard immunoassays?
Population
Stool specimens from human volunteers
Comparison
Reverse transcriptase-polymerase chain reaction… vs Radioimmunoassay, enzyme immunoassay, and…
Design
Other
Authors
Discussion
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Member takes
Implication
RT-PCR-OP may improve Norwalk virus stool detection; leaves open routine clinical adoption pending larger validation.
Key Points
- To develop and evaluate a sensitive, specific reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobe (RT-PCR-OP) method for detecting Norwalk virus in stool specimens.
- Amplified Norwalk virus genomic regions encoding RNA polymerase (260-bp) and a putative immunogenic protein (224-bp) using RT-PCR coupled with digoxigenin-labeled oligoprobe hybridization.
- Treated stool specimens with Sephadex G-200 gel chromatography to remove RT-PCR inhibitors.
- Evaluated specificity against other enteric viruses and validated the assay using 20 radioimmunoassay (RIA)-positive samples, 26 asymptomatic controls, and 21 coded specimens.
- Demonstrated a detection limit at least 100-fold greater than RIA, with zero cross-reactivity against human and animal caliciviruses, rotaviruses, enteroviruses, and other enteric pathogens.
- Detected Norwalk virus in 95% (19/20) of RIA-positive volunteer samples with immunogenic protein primers and 75% (15/20) with polymerase primers, while 26 asymptomatic controls yielded negative results.
- Identified Norwalk virus in 4 of 21 coded fecal specimens, matching the 4 samples positive by enzyme immunoassay.
Structured PICO
Does the RT-PCR-OP method accurately detect Norwalk virus in stool specimens compared to standard immunoassays?
P
Population
Stool specimens from human volunteers (20 positive for Norwalk virus by RIA, 26 from asymptomatic children and adults, and 21 coded fecal specimens)
I
Intervention
Reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobe (RT-PCR-OP) method
C
Comparator
Radioimmunoassay (RIA), enzyme immunoassay, and electron microscopy
O
Outcome
Detection of Norwalk virussurrogate
The developed RT-PCR-OP method provides a highly specific and sensitive approach for detecting Norwalk virus in stool specimens.
Limitations
- Two samples positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.
Cite This Study
Matsui et al. (1992) studied this question.