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Updated: Apr 13, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation
Yousry Hawash1, M M Ghonaim2, Ayman S Al-Hazmi3
1Department of Medical Parasitology, National Liver Institute, Menoufia University, Menoufia, Egypt ; Department of Medical Laboratory Science, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia.
Abstract:
Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.
Insights
This study developed an internal amplification control (IAC) to monitor PCR assays for Cryptosporidium in human feces, ensuring accurate negative results by preventing false negatives. The IAC reliably detected inhibition, confirming the validity of diagnostic PCR testing.
Area of Science:
- Molecular Biology
- Parasitology
- Clinical Diagnostics
Background:
- PCR assays for pathogen detection can be inhibited by substances in clinical samples like feces, leading to false-negative results.
- Ensuring the accuracy of negative PCR results requires a reliable inhibition control.
- Cryptosporidium detection in human feces is crucial for diagnosing parasitic infections.
Purpose of the Study:
- To develop and validate a competitive internal amplification control (IAC) for a PCR assay detecting Cryptosporidium in human feces.
- To ensure the reliability of diagnostic PCR by monitoring for inhibition.
- To assess the impact of the IAC on the analytical and diagnostic sensitivity of the PCR assay.
Main Methods:
- A cloning vector with a modified target DNA sequence was constructed using inverse PCR and restriction digestion.
- The modified DNA served as an internal amplification control (IAC) in a duplex PCR assay.
- The IAC was tested on fecal samples spiked with varying Cryptosporidium oocyst counts and on naturally infected samples.
Main Results:
- The developed IAC was successfully integrated into a PCR assay for Cryptosporidium detection in human feces.
- The IAC demonstrated effectiveness in monitoring PCR inhibition, with amplicons detected in all negative samples.
- Integration of the IAC did not compromise the analytical or diagnostic sensitivity of the PCR assay.
Conclusions:
- The constructed IAC is a valuable tool for validating PCR-based Cryptosporidium detection in human feces.
- The combined use of an optimized DNA extraction protocol and the IAC enhances the reliability of diagnostic PCR.
- This approach ensures the accuracy of negative results, crucial for effective patient management.
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