LIMIT OF MOLECULAR DETECTION OF STRONGYLOIDES STERCORALIS PARASITE PERFORMING TWO PCR PROTOCOLS | ||||
| Journal of the Egyptian Society of Parasitology | ||||
| Article 11, Volume 46, Issue 3, December 2016, Page 549-556 PDF (279.4 K) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/jesp.2016.88255 | ||||
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| Authors | ||||
| MOHAMED S. BADR1; HALA M. EL-ASKARY2; MARWA ELMALAWANY3; REHAM KAMAL3; NASHWA ALSAMRA4 | ||||
| 1Department of Microbiology and Molecular Biology, Faculty of Medicine, Ain Shams University, Egypt. | ||||
| 2Department of Medical Parasitology, Faculty of Medicine, Beni-Suef University, Egypt. | ||||
| 3Department of Medical Parasitology, Faculty of Medicine, Cairo University, Egypt. | ||||
| 4Department of Pediatric, Faculty of Medicine, Fayoum University, Egypt | ||||
| Abstract | ||||
| Low parasite density in chronic infection with S. stercoralis is a challenging diagnostic issue. Generally, molecular techniques don’t depend on parasite viability while copro-culture or Baermann concentration method relies on the presence of living larvae in fecal samples. Therefore, evaluation of PCR-based methods is important to increase the detection rates in light or chronic infection. This study was designed to analyze the sensitivity of quantitative PCR (qPCR) and nested PCR (nPCR) regarding the minimum amount of S. stercoralis DNA template that can be reliably detected by each molecular technique. Strongyloides larvae were collected from cultured stool samples from suspected infected Egyptian children. After counting larvae present in a known volume under the microscope, DNA extraction was done and serial dilution of genomic materials was prepared. Then, qPCR and nPCR targeting the small subunit of the rRNA gene were performed. Regarding qPCR, the limit of detection was 0.0005 S. stercoralis larvae/μl, with crossing threshold (Ct) value ranged from 17.8 to 38.7 while, nPCR did not detect from (0.002 to 0.00012 S. stercoralis larvae/μl) with minimum limit 0.004 S. stercoralis larvae/μl. Real-time quantitative PCR is very sensitive technique that can detect very low genomic load up to about 10 (9.765) genome copies/reaction compared to nested PCR which started positivity from 78.125 genome copies/reaction. Therefore, qPCR is recommended to detect chronic strongyloidiasis especially in high-risk groups to prevent lifethreatening spread of such infection. | ||||
| Keywords | ||||
| Strongyloides stercoralis; Limit of detection; Nested PCR; Quantitative PCR | ||||
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