Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection

Shaw Yi Kao, Tina M. Niemiec, Mike J. Loeffelholz, Beverly Dale, Harley A. Rotbart

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Abstract. Background: Enteroviruses (EV) cause a broad spectrum of human diseases, of which aseptic meningitis is among the most common and most clinically vexing. While the clinical symptoms of meningitis caused by bacteria, fungi and viruses are similar, the diagnosis, therapy and outcome of disease caused by these agents vary greatly. In order to appropriately manage meningitis patients, rapid and reliable diagnosis of EV meningitis impacts significantly on patient management. Objective: To develop a direct and uninterrupted RNA amplification of enteroviruses using rTth DNA polymerase. Study design: Purified coxsackievirus B6 RNA of various concentrations was amplified by rTth DNA polymerase-mediated amplification to determine analytic sensitivity. The specificity of the EV amplification was examined with a panel of nucleic acids from 36 EV serotypes, 15 non-EV pathogens and 10 coded clinical specimens of cerebrospinal fluid (CSF). Results: All EV serotypes tested were detected successfully by this method at a sensitivity of 1 TCID50 with the exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected at 10 TCID50, and echovirus 1 was detected at 100 TCID50. Echoviruses 22 and 23 were not detectable at 100 TCID50. Cross-reactivity of EV RT-PCR assay with 15 known non-EV meningitis pathogens has not been observed. Results of 10 CSF tested with this system correlated well with tissue culture. Conclusions: We have developed an EV amplification assay which has several important advantages over previously reported methods. This assay employs rTth DNA polymerase which possesses both reverse transcriptase and DNA polymerase activities, simplifying RNA reverse transcription and DNA amplification to an uninterrupted reaction. Additionally, potential carryover contamination and enhanced amplification specificity is provided by substituting dUTP for dTTP and adding uracil N-glycosylase (UNG) in the amplification reaction. Finally, the detection of amplified product is via a colorimetric, microwell format permitting the use of readily available instrumentation.

Original languageEnglish (US)
Pages (from-to)247-257
Number of pages11
JournalClinical and Diagnostic Virology
Volume3
Issue number3
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Enterovirus
RNA
DNA-Directed DNA Polymerase
Human Enterovirus B
Meningitis
Parechovirus
Cerebrospinal Fluid
Uracil-DNA Glycosidase
Aseptic Meningitis
RNA-Directed DNA Polymerase
Nucleic Acids
Reverse Transcription
Fungi
Viruses
Bacteria
Polymerase Chain Reaction
DNA

Keywords

  • Aseptic meningitis
  • Enterovirus
  • Meningitis
  • RNA amplification
  • RT-PCR
  • rTth DNA polymerase

ASJC Scopus subject areas

  • Virology

Cite this

Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection. / Kao, Shaw Yi; Niemiec, Tina M.; Loeffelholz, Mike J.; Dale, Beverly; Rotbart, Harley A.

In: Clinical and Diagnostic Virology, Vol. 3, No. 3, 1995, p. 247-257.

Research output: Contribution to journalArticle

Kao, Shaw Yi ; Niemiec, Tina M. ; Loeffelholz, Mike J. ; Dale, Beverly ; Rotbart, Harley A. / Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection. In: Clinical and Diagnostic Virology. 1995 ; Vol. 3, No. 3. pp. 247-257.
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AU - Kao, Shaw Yi

AU - Niemiec, Tina M.

AU - Loeffelholz, Mike J.

AU - Dale, Beverly

AU - Rotbart, Harley A.

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N2 - Abstract. Background: Enteroviruses (EV) cause a broad spectrum of human diseases, of which aseptic meningitis is among the most common and most clinically vexing. While the clinical symptoms of meningitis caused by bacteria, fungi and viruses are similar, the diagnosis, therapy and outcome of disease caused by these agents vary greatly. In order to appropriately manage meningitis patients, rapid and reliable diagnosis of EV meningitis impacts significantly on patient management. Objective: To develop a direct and uninterrupted RNA amplification of enteroviruses using rTth DNA polymerase. Study design: Purified coxsackievirus B6 RNA of various concentrations was amplified by rTth DNA polymerase-mediated amplification to determine analytic sensitivity. The specificity of the EV amplification was examined with a panel of nucleic acids from 36 EV serotypes, 15 non-EV pathogens and 10 coded clinical specimens of cerebrospinal fluid (CSF). Results: All EV serotypes tested were detected successfully by this method at a sensitivity of 1 TCID50 with the exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected at 10 TCID50, and echovirus 1 was detected at 100 TCID50. Echoviruses 22 and 23 were not detectable at 100 TCID50. Cross-reactivity of EV RT-PCR assay with 15 known non-EV meningitis pathogens has not been observed. Results of 10 CSF tested with this system correlated well with tissue culture. Conclusions: We have developed an EV amplification assay which has several important advantages over previously reported methods. This assay employs rTth DNA polymerase which possesses both reverse transcriptase and DNA polymerase activities, simplifying RNA reverse transcription and DNA amplification to an uninterrupted reaction. Additionally, potential carryover contamination and enhanced amplification specificity is provided by substituting dUTP for dTTP and adding uracil N-glycosylase (UNG) in the amplification reaction. Finally, the detection of amplified product is via a colorimetric, microwell format permitting the use of readily available instrumentation.

AB - Abstract. Background: Enteroviruses (EV) cause a broad spectrum of human diseases, of which aseptic meningitis is among the most common and most clinically vexing. While the clinical symptoms of meningitis caused by bacteria, fungi and viruses are similar, the diagnosis, therapy and outcome of disease caused by these agents vary greatly. In order to appropriately manage meningitis patients, rapid and reliable diagnosis of EV meningitis impacts significantly on patient management. Objective: To develop a direct and uninterrupted RNA amplification of enteroviruses using rTth DNA polymerase. Study design: Purified coxsackievirus B6 RNA of various concentrations was amplified by rTth DNA polymerase-mediated amplification to determine analytic sensitivity. The specificity of the EV amplification was examined with a panel of nucleic acids from 36 EV serotypes, 15 non-EV pathogens and 10 coded clinical specimens of cerebrospinal fluid (CSF). Results: All EV serotypes tested were detected successfully by this method at a sensitivity of 1 TCID50 with the exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected at 10 TCID50, and echovirus 1 was detected at 100 TCID50. Echoviruses 22 and 23 were not detectable at 100 TCID50. Cross-reactivity of EV RT-PCR assay with 15 known non-EV meningitis pathogens has not been observed. Results of 10 CSF tested with this system correlated well with tissue culture. Conclusions: We have developed an EV amplification assay which has several important advantages over previously reported methods. This assay employs rTth DNA polymerase which possesses both reverse transcriptase and DNA polymerase activities, simplifying RNA reverse transcription and DNA amplification to an uninterrupted reaction. Additionally, potential carryover contamination and enhanced amplification specificity is provided by substituting dUTP for dTTP and adding uracil N-glycosylase (UNG) in the amplification reaction. Finally, the detection of amplified product is via a colorimetric, microwell format permitting the use of readily available instrumentation.

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KW - RT-PCR

KW - rTth DNA polymerase

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