Relevance of Cryptosporidium parvum hsp70 mRNA amplification as a tool to discriminate between viable and dead oocysts.
Two mRNA extraction methods were compared in this study to clarify the discrepancies found between authors regarding the presence of mRNA in inactivated Cryptosporidium parvum oocysts. Cryptosporidium parvum heat shock protein 70 (hsp70) mRNA extraction was performed by using oligo(dT)20-labeled magnetic beads or by incubating oocyst lysates with DNase I. Significant differences in mRNA recovery rates between these 2 techniques were observed when working on inactivated oocysts. We consistently detected hsp70 mRNA in oocysts heated at 60 C for 30 min and oocysts incubated in 10% formalin for 2 hr when using DNase I in the mRNA extraction procedure. In contrast, no mRNA was detected in such oocysts when magnetic beads were used for the mRNA extraction. The selective capture of long poly-A tail mRNA, when using oligo(dT)20-labeled magnetic beads, is proposed in this paper for explaining the discrepancies observed between the two mRNA extraction methods compared in this study. DNA decay in inactivated and aging oocysts makes quantitative polymerase chain reaction a potential alternative technique for assessing C. parvum oocyst viability status in environmental samples.
CSIRO Land and Water, Wembley, WA, Australia.
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D000818:Animals; D002648:Child; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000254:growth & development*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan