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Updated: Jul 6, 2026
DNA Viral Size Fraction Metagenomics for Human Stool Samples
Published on: March 24, 2026
Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples
Eugenia Bolanaki1, Christine Kottaridi, Evaggelos Dedepsidis
1Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, University of Thessaly, 26 Ploutonos & Aiolou Street, Larisa 41221, Greece.
Abstract:
Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RT-PCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (subgrouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.
Insights
Routine diagnosis of acute flaccid paralysis (AFP) relies on cell culture, but RT-PCR offers superior detection of enteroviruses. This study highlights RT-PCR
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- Classical virological procedures for diagnosing acute flaccid paralysis (AFP) face challenges with enterovirus isolation.
- Standard cell culture methods often require multiple passages, potentially altering viral composition.
- Stool specimens present challenges for viral RNA extraction due to inhibitors and sample heterogeneity.
Purpose of the Study:
- To evaluate and compare RNA extraction methods for enterovirus detection in fecal samples.
- To assess the efficacy of RT-PCR compared to cell culture for enterovirus diagnosis.
- To establish a rapid and sensitive molecular method for enterovirus recovery and characterization.
Main Methods:
- Compared three RNA extraction methods, selecting the QIAamp Viral RNA Mini Kit.
- Analyzed 54 archived fecal samples using both cell culture and RT-PCR.
- Employed RT-PCR targeting the 5'NCR and VP1 regions for enterovirus detection and serotyping.
Main Results:
- The QIAamp Viral RNA Mini Kit provided high viral RNA yield without RT-PCR inhibitors.
- RT-PCR demonstrated a higher detection frequency for enteroviruses than cell culture.
- RT-PCR identified two additional positive samples missed by standard cell culture methods.
Conclusions:
- RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR is a rapid and sensitive technique.
- This molecular approach enables direct recovery and characterization of poliovirus and non-polio enteroviruses from fecal specimens.
- The method avoids cell culture passages, preserving the original viral genetic diversity for accurate diagnosis.
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