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Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection
S Y Kao1, T M Niemiec, M J Loeffelholz
1Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501, USA.
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 TCID(50) with the exception of echoviruses 1, 5, 22 and 23. Echovirus 5 was detected at 10 TCID(50), and echovirus 1 was detected at 100 TCID(50). Echoviruses 22 and 23 were not detectable at 100 TCID(50). 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.
Insights
A new assay rapidly detects enteroviruses (EV) causing meningitis. This method uses a single enzyme for RNA amplification, improving speed and specificity for faster diagnosis and patient management.
Area of Science:
- Molecular Biology
- Virology
- Clinical Diagnostics
Background:
- Enteroviruses (EV) are common causes of aseptic meningitis, a condition with similar symptoms to other meningitis types.
- Accurate and rapid diagnosis of EV meningitis is crucial for effective patient management.
Purpose of the Study:
- To develop a direct and uninterrupted RNA amplification assay for enteroviruses (EV) using rTth DNA polymerase.
- To improve the speed and specificity of EV detection in clinical samples.
Main Methods:
- rTth DNA polymerase was used to amplify purified coxsackievirus B6 RNA to determine assay sensitivity.
- Specificity was tested against 36 EV serotypes and 15 non-EV pathogens.
- The assay was validated using 10 cerebrospinal fluid (CSF) specimens.
Main Results:
- The assay detected most EV serotypes at a sensitivity of 1 TCID50, with minor exceptions.
- No cross-reactivity was observed with common non-EV meningitis pathogens.
- Results from CSF specimens correlated well with traditional tissue culture methods.
Conclusions:
- A novel EV amplification assay was developed, simplifying RNA reverse transcription and DNA amplification into a single, uninterrupted reaction.
- The use of dUTP and uracil N-glycosylase (UNG) enhances specificity and prevents contamination.
- The colorimetric microwell format allows for detection using standard laboratory equipment, facilitating clinical application.
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