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

Hawash Y, Ghonaim MM, Al-Hazmi AS - Korean J. Parasitol. (2015)

Bottom Line: When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus

Ethidium bromide-stained 1% agarose gel showing amplification products of the PCR assay using a number of Cryptosporidium-positive control fecal samples. M, GeneRulerTM 100 bp DNA marker; Lanes 1-4, 4 Cryptosporidium-positive samples; Lane 5, Cryptosporidium-negative sample; Lane 6, EAC (PCR positive control); Lane 7, no-template master mix sample (PCR negative control).
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f4-kjp-53-2-147: Ethidium bromide-stained 1% agarose gel showing amplification products of the PCR assay using a number of Cryptosporidium-positive control fecal samples. M, GeneRulerTM 100 bp DNA marker; Lanes 1-4, 4 Cryptosporidium-positive samples; Lane 5, Cryptosporidium-negative sample; Lane 6, EAC (PCR positive control); Lane 7, no-template master mix sample (PCR negative control).

Mentions: The Cryptosporidium native target DNA was successfully amplified, side by side with the IAC, in all Cryptosporidium-positive control samples (n=25), except 1 sample. This sample with false negative result showed IAC amplicon on gel and was 1 of the 3 samples with low oocysts density. Interestingly, the intensity of IAC (≈375 bp) and the native (≈550 bp) PCR products alternately appeared abnormally faint on gel in 2-3 samples (Fig. 4). None of the Cryptosporidium-negative control samples (n=45) showed amplification of the native COWP gene by the PCR assay. However, the IAC amplification products were successfully detected on gel for all samples. On view of these findings, the PCR assay was found to exhibit sensitivity, specificity, negative predictive value and positive predictive value of 96%, 100%, 98%, and 100%, respectively. Equally important, none of control samples DNA extracts showed detectable inhibition for the PCR amplifications.


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

Hawash Y, Ghonaim MM, Al-Hazmi AS - Korean J. Parasitol. (2015)

Ethidium bromide-stained 1% agarose gel showing amplification products of the PCR assay using a number of Cryptosporidium-positive control fecal samples. M, GeneRulerTM 100 bp DNA marker; Lanes 1-4, 4 Cryptosporidium-positive samples; Lane 5, Cryptosporidium-negative sample; Lane 6, EAC (PCR positive control); Lane 7, no-template master mix sample (PCR negative control).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4416379&req=5

f4-kjp-53-2-147: Ethidium bromide-stained 1% agarose gel showing amplification products of the PCR assay using a number of Cryptosporidium-positive control fecal samples. M, GeneRulerTM 100 bp DNA marker; Lanes 1-4, 4 Cryptosporidium-positive samples; Lane 5, Cryptosporidium-negative sample; Lane 6, EAC (PCR positive control); Lane 7, no-template master mix sample (PCR negative control).
Mentions: The Cryptosporidium native target DNA was successfully amplified, side by side with the IAC, in all Cryptosporidium-positive control samples (n=25), except 1 sample. This sample with false negative result showed IAC amplicon on gel and was 1 of the 3 samples with low oocysts density. Interestingly, the intensity of IAC (≈375 bp) and the native (≈550 bp) PCR products alternately appeared abnormally faint on gel in 2-3 samples (Fig. 4). None of the Cryptosporidium-negative control samples (n=45) showed amplification of the native COWP gene by the PCR assay. However, the IAC amplification products were successfully detected on gel for all samples. On view of these findings, the PCR assay was found to exhibit sensitivity, specificity, negative predictive value and positive predictive value of 96%, 100%, 98%, and 100%, respectively. Equally important, none of control samples DNA extracts showed detectable inhibition for the PCR amplifications.

Bottom Line: When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection.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.

View Article: PubMed Central - PubMed

Affiliation: Department 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.

No MeSH data available.


Related in: MedlinePlus