Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler’s Diarrhea
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
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.
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.
참고문헌 (30)
- Diemert DJ, "Prevention and self-treatment of traveler's diarrhea", Clin Microbiol Rev, 19, pp.583~594 (2006)
- DuPont HL, "Travellers’ diarrhoea : contemporary approaches to therapy and prevention", Drugs, 66, pp.303~314 (2006)
- Wood AJ, "Prevention and treatment of traveler's diarrhea", New Engl J Med, 328, pp.1821~1827 (1993)
- Bradley AC, "CDC Health Information for International Travel 2016" (2016), Oxford University Press
- Freedman DO, "Spectrum of disease and relation to place of exposure among ill returned travelers", New Engl J Med, 354, pp.119~130 (2006)
- Baldursson S, "Waterborne transmission of protozoan parasites : review of worldwide outbreaks–an update 2004-2010", Water Res, 45, pp.6603~6614 (2011)
- Strausbaugh LJ, "Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s", Clin Infect Dis, 31, pp.1040~1057 (2000)
- Jae-RanYu, "A Case of Human Cyclosporiasis Causing Traveler’s Diarrhea after Visiting Indonesia", Journal of Korean Medical Science, 18(5), pp.738~741 (2003), 대한의학회
- Don R, "'Touchdown' PCR to circumvent spurious priming during gene amplification", Nucleic Acids Res, 19, pp.4008~ (1991)
- 유재란, "Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum", The Korean Journal of Parasitology, 47(3), pp.293~297 (2009), 대한기생충학ㆍ열대의학회
- Cheun HI, "Development of a diagnostic kit to detect Cryptosporidium parvum and Giardia lamblia", Osong Public Health Res Perspect, 4, pp.146~151 (2013)
- Shirley M, "The genome of Eimeria spp., with special reference to Eimeria tenella-a coccidium from the chicken", Int J Parasitol, 30, pp.485~493 (2000)
- Guay JM, "Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii : primary and secondary structure of the 5S gene", Gene, 114, pp.165~171 (1992)
- Spano F, "PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin", FEMS Microbiol Lett, 150, pp.209~217 (1997)
- Ericsson CD, "Traveler's diarrhea due to intestinal protozoa", Clin Infect Dis, 33, pp.110~114 (2001)
- Graczyk TK, "Human waterborne trematode and protozoan infections", Adv Parasitol, 64, pp.111~160 (2007)
- Gajadhar AA, "Factors contributing to the public health and economic importance of waterborne zoonotic parasites", Vet Parasitol, 126, pp.3~14 (2004)
- Ramírez-Castillo FY, "Waterborne pathogens : detection methods and challenges", Pathogens, 4, pp.307~334 (2015)
- Law JWF, "Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations", Front Microbiol, 5, pp.770~ (2015)
- Verweij JJ, "Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR", J Clin Microbiol, 42, pp.1220~1223 (2004)
- Haque R, "Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp", Am J Trop Med Hyg, 76, pp.713~717 (2007)
- Li W, "A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis", Vet Parasitol, 173, pp.11~18 (2010)
- Pierce KK, "Update on human infections caused by intestinal protozoa", Curr Opin Gastroenterol, 25, pp.12~17 (2009)
- Kansouzidou A, "Cyclospora cayetanensis in a patient with travelers' diarrhea : case report and review", J Travel Med, 11, pp.61~63 (2004)
- Lalonde LF, "Highly sensitive and specific PCR assay for reliable detection of Cyclospora cayetanensis oocysts", Appl Environ Microbiol, 74, pp.4354~4358 (2008)
- Read CM, "Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP", Infect Genet Evol, 4, pp.125~130 (2004)
- Fayer R, "Cryptosporidium and cryptosporidiosis", pp.1~41 (1997), CRC Press Inc
- Ghosh S, "PCR detection of Giardia lamblia in stool : targeting intergenic spacer region of multicopy rRNA gene", Mol Cell Probes, 14, pp.181~189 (2000)
- David E, "Diagnosis of Giardia infections by PCR-based methods in children of an endemic area", J Venom Anim Toxins, 17, pp.209~215 (2011)
- Orlandi PA, "Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR", Appl Environ Microb, 69, pp.4806~4813 (2003)