Simple method for long-term copro-preservation of Cryptosporidium oocysts for morphometric and molecular analysis.
Preservation of Cryptosporidium oocysts in faecal specimens containing 75% ethanol is suitable for subsequent morphometric and molecular analysis. No significant morphologic alteration occurred after storage at ambient temperatures, ranging from 22 to 38 degrees C, for more than 2 years. After washing, sugar floatation and DNA extraction, a nested polymerase chain reaction targeting the small subunit ribosomal RNA gene successfully amplified Cryptosporidium DNA in all 15 isolates examined. The sensitivity of detection by polymerase chain reaction (PCR) was found to be as high as 1.25 oocysts per reaction (mean=3.01, SD=1.14). Importantly, a 2.2-kb of the complete DNA sequence of a gene encoding Cryptosporidium thrombospondin-related adhesive protein (TRAP-C1) was also consistently amplified by PCR in all isolates. The PCR-amplified product can be used as a good template for sequencing. Therefore, this simple procedure should be useful for epidemiological analysis of clinical samples from outbreaks, endemic or sporadic cases of cryptosporidiosis when long-term storage of oocysts is required.
Jongwutiwes
Somchai
S
Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. fmedsjw@md2.md.chula.ac.th
Tiangtip
Rattana
R
Yentakarm
Sutin
S
Chantachum
Nutaros
N
D000818:Animals; D003458:Cryptosporidium / Q000235:genetics*; D000431:Ethanol; D005243:Feces / Q000469:parasitology*; D021141:Nucleic Acid Amplification Techniques; D019188:Organ Preservation Solutions; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors
D019188:Organ Preservation Solutions; D012335:RNA, Ribosomal; D000431:Ethanol