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Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic
J Petitjean1, M Quibriac, F Freymuth
1Laboratoire de Virologie, Centre Hospitalier Régional Universitaire de Caen, France.
Abstract:
Enteroviruses were specifically detected in crude clinical specimens or in cell cultures in which the viruses were amplified by dot hybridization by using poliovirus type 1-derived, subgenomic radiolabeled cRNA probes (riboprobes). The sensitivity of this test varied from 2.5 to 33%, when clinical specimens without cell culture were examined, and was about 85% in cell culture lysates. The specificity of the test was 90 to 100%. The riboprobe corresponding to the 5'-noncoding sequence specifically detected the majority of enteroviruses (56 of 57 tested); the riboprobe derived from the VPI capsid region hybridized with the three poliovirus serotypes and with some coxsackieviruses type A and with echovirus type 7. Echovirus 22 did not hybridize with any riboprobe. In stool specimens, nasal aspirates, and cerebrospinal fluids from patients with meningitis, only one type of virus was identified in different clinical samples from the same patient by the seroneutralization test. Hybridization allowed the detection of enteroviral RNAs easily in stool specimens and nasal aspirates but with a low efficiency in cerebrospinal fluids without amplification of the viruses in cell cultures.
Insights
This study developed a sensitive dot hybridization test using riboprobes for detecting enteroviruses in clinical samples. The method showed high specificity and detected most enteroviruses, aiding in viral diagnosis.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- Enteroviruses are a significant cause of human illness.
- Accurate and rapid detection of enteroviruses is crucial for diagnosis and treatment.
- Current diagnostic methods can be time-consuming or lack sensitivity.
Purpose of the Study:
- To develop and evaluate a dot hybridization assay for enterovirus detection.
- To assess the sensitivity and specificity of this assay using riboprobes.
- To compare the performance of the assay in crude clinical specimens versus cell cultures.
Main Methods:
- Dot hybridization using radiolabeled subgenomic cRNA probes (riboprobes) derived from poliovirus type 1.
- Probes targeted the 5'-noncoding sequence and the VPI capsid region.
- Testing on crude clinical specimens (stool, nasal aspirates, cerebrospinal fluid) and cell culture lysates.
- Comparison with seroneutralization tests.
Main Results:
- The assay demonstrated 90-100% specificity.
- Sensitivity varied (2.5-33% in crude specimens, 85% in cell cultures).
- A 5'-noncoding sequence probe detected most enteroviruses (56/57); a VPI probe detected polioviruses and some coxsackieviruses/echoviruses.
- Enteroviral RNA was detected efficiently in stool and nasal aspirates but poorly in cerebrospinal fluid without cell culture amplification.
Conclusions:
- Dot hybridization with specific riboprobes is a viable method for enterovirus detection.
- Amplification in cell culture significantly enhances assay sensitivity.
- The 5'-noncoding sequence probe offers broad enterovirus detection capabilities.
- The assay is most effective for stool and nasal specimens, with limitations for cerebrospinal fluid without prior virus amplification.