Korean J Parasitol.  2016 Oct;54(5):631-636. 10.3347/kjp.2016.54.5.631.

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea

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

• KMID: 2360527
• DOI: http://doi.org/10.3347/kjp.2016.54.5.631

Abstract

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Keyword

Cryptosporidium parvum; Giardia lamblia; Cyclospora cayetanensis; multiplex PCR; touchdown PCR; stool sample

MeSH Terms

Cryptosporidium parvum*
Cryptosporidium*
Cyclospora*
Diarrhea*
Genes, rRNA
Giardia lamblia*
Giardia*
Glutamate Dehydrogenase
Humans
Methods
Multiplex Polymerase Chain Reaction
Oocysts
Parasites
Polymerase Chain Reaction*
RNA, Ribosomal, 18S
Glutamate Dehydrogenase
RNA, Ribosomal, 18S