Study of 18S rRNA and rDNA stability by real-time RT-PCR in heat-inactivated Cryptosporidium parvum oocysts.
The public health problem posed by Cryptosporidium parvum has led the water supply industry to develop analytical tools for detecting viable oocysts in water. In this study, we report on a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) method that targets and quantifies C. parvum 18S rRNA. To study the suitability of 18S rRNA as an indicator of Cryptosporidium oocyst viability, the stability of 18S rRNA and rDNA was monitored by real-time RT-PCR following various Cryptosporidium heat treatments. Decay of 18S rRNA was first observed after a 20-min treatment of C. parvum oocysts at 95 degrees C and was still detectable after 4 h. In contrast, rDNA was more heat resistant. The stability of 18S rRNA and rDNA was also studied after oocyst lysis by thermal shocks in the presence and absence of Chelex-100. In the former case, both rRNA and rDNA were degraded whereas in the presence of Chelex-100 both molecules were protected from heat degradation and were still detected after 4 h at 95 degrees C following thermal shocks. Our results indicate that 18S rRNA detection may not be directly associated with viability following heat inactivation of Cryptosporidium oocysts even if in all the experiments 18S rRNA was less stable than rDNA.
Fontaine
Melanie
M
Suez-Environnement Local Services, CIRSEE, 38 Avenue du Président Wilson, 78230 Le Pecq, France. fontainemelanie@voila.fr
Guillot
Emmanuelle
E
D000818:Animals; D002614:Chelating Agents; D016785:Cryptosporidium parvum / Q000502:physiology*; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000032:analysis; D004203:Disinfection / Q000379:methods; D020459:Genes, rRNA; D006358:Hot Temperature; D033661:Oocysts / Q000502:physiology*; D016053:RNA, Protozoan / Q000032:analysis / Q000302:isolation & purification; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012117:Resins, Synthetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014867:Water / Q000469:parasitology
D002614:Chelating Agents; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D012117:Resins, Synthetic; D014867:Water; C024997:Chelex 100