The Impact of Different Copro-preservation Conditions on Molecular Detection of Cryptosporidium Species
Background: We aimed to evaluate different copro-preservation conditions along the duration of one month for a better outcome of molecular diagnosis of Cryptosporidium species.
Methods: Ten samples out of 380 fresh stool samples collected from patients with diarrhea proved positive after direct examination, concentration, staining and confirmed by immunochromatographic test. The study was conducted at the Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University at the time interval from July 2014 to December 2015. Each stool sample was preserved in five different conditions; freezing at -20 ºC, 70% ethyl alcohol, 10% formalin, 2.5% potassium dichromate (K dichromate) at 4 ºC and 2.5% K dichromate at room temperature (RT). Then DNA extraction and nested PCR, with Cryptosporidium oocyst wall protein (COWP) gene were done from each sample at zero time (fresh specimens) as a standard for comparison with the preservation conditions at 10, 20 and 30 d.
Results: Sensitivity of studied preservative conditions along the whole study duration showed best outcome from freezing at -20 °C (80%) then K dichromate (4 °C) (73.3%) followed by K dichromate (RT) (66.7%), then alcohol (33.3%), while formalin was the worst (0%) with a highly significant comparative outcome between the different conditions. Along the three extraction intervals, K dichromate (RT), unlike all the rest of conditions lacks the consistent preservative action.Conclusion: Our study highlights freezing at -20 ºC to be the most suitable condition for preservation followed by K dichromate at 4 °C, K dichromate at RT, then 70% ethyl alcohol. Formalin (10%) is better to be avoided.
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|Issue||Vol 12 No 2 (2017)|
|Cryptosporidium Stool examination Molecular diagnosis Fecal preservatives Diarrhea Nested PCR|
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