A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.
BACKGROUND
Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable.


OBJECTIVE
To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination.


METHODS
The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel.


RESULTS
Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2.


CONCLUSIONS
The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory.
Mohamed
Nahla
N
Section of Virology, Department of Medical Sciences, Uppsala University, Uppsala 75185, Sweden.
Elfaitouri
Amal
A
Fohlman
Jan
J
Friman
Göran
G
Blomberg
Jonas
J
D020121:5' Untranslated Regions / Q000235:genetics; D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000134:cerebrospinal fluid* / Q000175:diagnosis; D006801:Humans; D008587:Meningitis, Viral / Q000134:cerebrospinal fluid / Q000821:virology; D008875:Middle Aged; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000134:cerebrospinal fluid* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D020121:5' Untranslated Regions; D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic