title: Evaluation of two methods for quantification of hsp70 mRNA from the waterborne pathogen Cryptosporidium parvum by reverse transcription real-time PCR in environmental samples.
abstract: We optimized and evaluated two mRNA extraction methods to quantify induced hsp70 mRNA from viable and injured Cryptosporidium parvum oocysts by reverse transcription quantitative real-time PCR (RT-qPCR) in raw and treated manure. Methods based on guanidinium isothiocyanate/phenol/chloroform (GITC-PC) purification and direct mRNA extraction with magnetic oligo(dT)25-coated beads were evaluated for applicability and sensitivity. Both methods proved to be suitable for processing manure samples. With washed manure samples and oocyst disruption by bead beating for 165 s in time intervals with cumulative pooling of the lysate fractions, optimum RT-qPCR results were achieved. On average, 2.6 times more hsp70 mRNA was detected with the oligo(dT)25 method in comparison to the GITC-PC based method using fresh oocysts, whereas less mRNA was detected in aged oocysts. For fresh oocysts, analytical and method detection limits for the oligo(dT)25 based method were 1.7 cDNA copies/qPCR reaction and 5150 oocysts/mL manure, and for the GITC-PC based method 17 cDNA copies/qPCR reaction and 4950 oocysts/mL, respectively. In 12 months old oocysts with reduced viability, mRNA was occasionally detected only by the GITC-PC based method. Failure of or reduced detection with the oligo(dT)25 based method was apparently a result of weakened oocyst walls leading to quicker release of mRNA and therefore mRNA shredding by bead beating in the relatively long stretch between the capture sequence and the RT-qPCR target sites.
authors.affiliation: Institute of Water Quality Control, Technische Universität München, Am Coulombwall, 85748 Garching, Germany. g_garces@gmx.net
authors.forename: Gabriela
authors.initials: G
authors.lastname: Garcés-Sanchez
authors.forename: Peter A
authors.initials: PA
authors.lastname: Wilderer
authors.forename: Jean Charles
authors.initials: JC
authors.lastname: Munch
authors.forename: Harald
authors.initials: H
authors.lastname: Horn
authors.forename: Michael
authors.initials: M
authors.lastname: Lebuhn
mesh_terms: D000818:Animals; D002725:Chloroform / Q000737:chemistry; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000302:isolation & purification; D006146:Guanidines / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D033661:Oocysts / Q000378:metabolism*; D019800:Phenol / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013861:Thiocyanates / Q000737:chemistry
chemical_list: D006146:Guanidines; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D013861:Thiocyanates; D019800:Phenol; C054436:guanidine thiocyanate; D002725:Chloroform