Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples.
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.
Bolanaki
Eugenia
E
Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, University of Thessaly, 26 Ploutonos & Aiolou Street, Larisa 41221, Greece.
Kottaridi
Christine
C
Dedepsidis
Evaggelos
E
Kyriakopoulou
Zaharoula
Z
Pliaka
Vaia
V
Pratti
Anastassia
A
Levidiotou-Stefanou
Stamatina
S
Markoulatos
Panayotis
P
D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D005243:Feces / Q000821:virology*; D016679:Genome, Viral; D006801:Humans; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral