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Development of internal controls for probe-based nucleic acid diagnostic assays
B C Courtney1, M M Smith, E A Henchal
1Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011, USA.
Analytical Biochemistry
|May 21, 1999
Summary
This study introduces a novel internal control for nucleic acid diagnostic assays, enhancing accuracy. The method distinguishes true negatives from false negatives caused by reaction inhibition in PCR-ELISA.
Area of Science:
- Molecular Biology
- Diagnostic Assay Development
- Microbiology
Background:
- Probe-based nucleic acid diagnostic assays are crucial for pathogen detection.
- Ensuring assay reliability requires robust internal controls to differentiate true negatives from false negatives.
- Existing methods may not adequately address reaction inhibition or assay failure.
Purpose of the Study:
- To design, evaluate, and apply a novel internal control system for probe-based nucleic acid diagnostic assays.
- To develop a method for differentiating true negative results from false negative results.
- To improve the reliability and accuracy of diagnostic assays, such as PCR-ELISA.
Main Methods:
- Utilized oligonucleotide-directed mutagenesis and PCR amplification to create a modified probe-annealing sequence.
- Developed an internal control target within a cloned IS1111a gene fragment of Coxiella burnetii.
- Designed internal control probes with specific annealing characteristics distinct from wild-type probes.
Main Results:
- The internal control probe-recognition site shared key characteristics (length, base composition, location, annealing temperature) with the wild-type probe.
- Crucially, internal control and wild-type probes did not anneal to each other's recognition sequences.
- Amplified internal control and wild-type nucleic acid fragments were identical in length and base composition, not affecting amplification conditions.
- Small quantities of the internal control clone could be added without negatively impacting the assay.
- Successfully differentiated true negative results from false negative results in a single reaction.
Conclusions:
- The developed internal control system effectively validates nucleic acid diagnostic assays.
- This method enhances the ability to distinguish between the absence of pathogen genetic material and assay failure.
- The technique offers a reliable solution for improving the accuracy of probe-based diagnostic tests.