Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation
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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.
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.
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