Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR
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Key result
A modified hepatitis delta virus RNA internal control implemented in multiplex RT-PCR assays did not compromise detection limits and successfully distinguished genuine from false-negative results.
Why the study?
Does a modified HDV RNA internal control improve diagnostic confidence without compromising detection limits in multiplex RT-PCR for respiratory viruses?
Population
324 consecutive unselected respiratory samples and 191 archived known positive samples for respiratory viruses
Comparison
Modified hepatitis delta virus RNA internal… vs RT-PCR without the internal control
Design
Other
Authors
Discussion
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Implication
May distinguish false negatives in multiplex RT-PCR without sensitivity loss; hypothesis-generating pending prospective validation.
Structured PICO
Does a modified HDV RNA internal control improve diagnostic confidence without compromising detection limits in multiplex RT-PCR for respiratory viruses?
P
Population
324 consecutive unselected respiratory samples tested for nine clinically important respiratory viruses to validate a novel RNA internal control.
I
Intervention
Modified hepatitis delta virus (HDV) RNA internal control implemented in three nested multiplex RT-PCRs
C
Comparator
RT-PCR without the internal control
O
Outcome
Limit of detection and detection of PCR inhibitionsurrogate
A modified HDV RNA internal control can be effectively used in routine multiplex RT-PCR for respiratory viruses without compromising assay sensitivity, improving diagnostic confidence by identifying false negatives.
Limitations
- The control sequence may rarely be present in certain clinical samples, such as blood from patients coinfected with HBV and HDV.
- Multiplexing with highly degenerate primers can cause the control amplicon to be amplified in preference to the virus amplicon at low target concentrations.
- The high G+C content of the HDV control may adversely affect all rounds of the PCR.
- Lower limit of detection at the viral RNA level was not determined
- Control sequence may rarely be present in certain samples like blood from HBV/HDV coinfected patients
Cite This Study
Dingle et al. (2004) studied Respiratory viral infections (n=324). Modified hepatitis delta virus (HDV) RNA internal control vs. Standard RT-PCR without internal control was evaluated on Assay detection limit and identification of PCR inhibition. A modified hepatitis delta virus RNA internal control implemented in multiplex RT-PCR assays did not compromise detection limits and successfully distinguished genuine from false-negative results.
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