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Original Article

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

The Korean Journal of Parasitology 2018;56(5):419-427.
Published online: October 31, 2018

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*Corresponding author (ehshin@snu.ac.kr)
• Received: August 20, 2018   • Revised: September 20, 2018   • Accepted: September 21, 2018

Copyright © 2018 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples
Korean J Parasitol. 2018;56(5):419-427.   Published online October 31, 2018
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Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples
Korean J Parasitol. 2018;56(5):419-427.   Published online October 31, 2018
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Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples
Image Image Image Image
Fig. 1 Amplification by real-time PCR signals on C. parvum (Cp), G. lamblia (Gl), C. cayetanensis (Cc), and bacteriophage T4 (BpT4) chosen target genes using a primer-probe cocktail. The mixture of 4 primer pairs and 4 kinds of probes was prepared in advance and reacted with the DNA mixture of Cp, Gl, Cc, and Bp using the quadroplex real-time PCR method. Fluorescent dyes used in present study were as follows. Cp (blue line): FAM™; Gl (green line): HEX™; Cc (purple line): Cy5™; Bp (red line): CAL Fluor Red® 610.
Fig. 2 Standard curve of protozoa genomic DNA amplification by real-time PCR using a primer-probe cocktail. The 10-fold serial diluted (103, 104, 105, and 106) genomic DNA was extracted from oocyst of Cp (A) and cyst of Gl (B). The 10-fold serial diluted concentration (106, 105, 104, and 103) genomic DNAs of Cc (C) and Bp (D) were prepared from purchased Cc DNA and T4 phage, respectively. Cp (blue line): FAM™; Gl (green line): HEX™; Cc (purple line): Cy5™; Bp (T4 phage, red line): CAL Fluor Red® 610.
Fig. 3 Limit of detection (LOD) for each parasite in parasite-spiked human stool samples using the multiplex real-time PCR assay method. LOD of each parasite was investigated with a primer-probe cocktail and determined by Ct values. BpT4 (107 CFU) used as an internal control were also spiked with each parasite in the stool sample. (A) Cp (blue line) and Bp (red line). (B) Gl (green line) and Bp (red line). (C) Cc (purple line) and Bp (red line).
Fig. 4 Simultaneous detection of Cp, Gl, and Cc in the co-spiked human stool sample using our multiplex real-time PCR assay method. Amplification curves for each parasite in the co-spiked stool sample (Cp, Gl, and Cc) was confirmed by the line colored blue (FAM™), green (HEX™), and purple (Cy5™), respectively.
Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

Sequences of primers and probes used for multiplex real-time PCR for C. parvum, G. lamblia and C. cayetanensis, and internal control T4 phage target genes

Target parasite Primer/Probe Direction Reporter/Quencher Sequence (5′-3′) Target GenBank accession no.
Cryptosporidium parvum Cp-RT-F F CAA GGC CTC CAA TGT ATA CAA AC Cryptosporidium oocyst wall protein (COWP) AB089292
Cp-RT-R R CGA TTG CTT GGA CTG AAA TAC AG
Cp-RT-Pro FAM/BHQ-1® CCT GTC TGC CCT CCA GGT ACA GTA TTA C

Giardia lamblia Gl-RT-F F CTG AAG AAC TCC CTC ACC AC Glutamate dehydrogenase (GDH) KJ499992
Gl-RT-R R CAG AAG CGC ATG ACC TCG TTG
Gl-RT-Pro HEX/BHQ-1® CAA GGG CGG CTC CGA CTT TGA CCC AA

Cyclospora cayetanensis Cc-RT-F F GGT TTA TTG CTG CTG GCA GTT C Internal transcribed spacer 1 (ITS 1) AF302546
Cc-RT-R R CAA CAC CAC CAC AGA CAC GTG
Cc-RT-Pro Cy5/BHQ-2® GTG GTG GTG TTG CAG CAG TGG TGT TC

Enterobacteria phage T4 Bp-RT-F F CAA GCT CTC GGA GAA TTA AAT CAT Gene product 21 (gp21) M15359
Bp-RT-R R GAA CCT AAT CCA CGA GAA GAA ACT
Bp-RT-Pro CAL Fluor Red® 610/BHQ-2® GAC GAG CTC GTG TTA TTG AAG GTG ACC A
Table 1 Sequences of primers and probes used for multiplex real-time PCR for C. parvum, G. lamblia and C. cayetanensis, and internal control T4 phage target genes