Korean J Parasitol.  2018 Oct;56(5):419-427. 10.3347/kjp.2018.56.5.419.

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

• Shin JH
• Lee SE
• Kim TS
• Ma DW
• Cho SH
• Chai JY
• Shin EH

• KMID: 2426746
• DOI: http://doi.org/10.3347/kjp.2018.56.5.419

Abstract

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAMâ„¢, HEXâ„¢, Cy5â„¢, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

Keyword

Cryptosporidium parvum; Giardia lamblia; Cyclospora cayetanensis; traveler's diarrhea; bacteriophage T4; multiplex PCR; real-time PCR; Taq-Man assay; internal control

MeSH Terms

Bacteriophage T4*
Cryptosporidium parvum*
Cryptosporidium*
Cyclospora*
Diagnosis*
Diarrhea
DNA
Fluorescent Dyes
Giardia lamblia*
Giardia*
Glutamate Dehydrogenase
Humans*
Limit of Detection
Methods
Multiplex Polymerase Chain Reaction
Oocysts
Parasites
Real-Time Polymerase Chain Reaction*
DNA
Fluorescent Dyes
Glutamate Dehydrogenase