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the primers for the pcr reaction were designed using mit bioinformatics package primer3 version 4.0.0 [ 31 ] and optimized using gradient pcr amplifications and real-time pcr validation of each primer pair against various stool samples from zanzibar, haiti, and africa. real-time pcr reactions were carried out in separate, 96-well optical pcr plates using a rotor-gene 6000 real-time pcr machine (corbett life science, sydney, nsw, australia). a 10 ul reaction comprised of 5 ul of dna and 5 ul of the master mix. the 10 ul master mix contained: 0.5 ul of eva green dye; 0.75 ul of 10x primer/probe mix; 0.15 ul of rox; and 0.25 ul of forward and reverse primers (5 um). cycling was carried out at a ramp rate of 42.5c for 10 minutes and then heated to 95c for 10 minutes. cycling was then performed at a ramp rate of 1.6c for 60 seconds, followed by 55c for 1 minute, with cycles repeated for 40 times. the results were analyzed with the rotor-gene 6000 software. standard curves were created using serial dilutions of the plasmid with the nuclear expression vector pcdna3.1, and used to construct the pcr efficiency (e) and correlation coefficient (r 2 ). s. japonicum positive and negative control samples were supplied by the national veterinary services laboratory, usa and the national museum of the united states, respectively, and positive and negative s. mansoni and s. haematobium control samples were also provided by the filariasis research reagent resource center, university of georgia, athens, ga. all standards and samples were tested in duplicate.
the qpcr assay for s. mansoni designed in this study was compared with a published reference assay described by marcus et al. and cestari et al. [ 17, 30 ]. briefly, an s. mansoni positive and negative control sample were provided by the filariasis research reagent resource center and a sample from a gambian patient was provided by dr. david lejon, geneva, switzerland. additional control samples from zanzibar (s. mansoni), culex pipiens, and anopheles stephensi were kindly provided by dr. john werre, armauer hansen research institute and dr. paolo montalto, istituto superiore di sanità, rome, italy. samples from the three mosquito species were extracted with the phenol-chloroform method. the protocol used for mosquito extraction followed the manufacturer protocol from the qiagen dneasy blood & tissue kit (qiagen, germantown, md). extractions were performed in duplicate for each sample. for each extraction, a negative control was included in each run to confirm the absence of contamination. the qpcr was performed in 96-well optical pcr plates, in triplicate, using the primer/probe assays developed in this study and the published assays. amplification was performed in separate, 96-well optical pcr plates using a rotor-gene 6000 real-time pcr machine (corbett life science, sydney, nsw, australia). a 10 ul reaction comprised of 5 ul of dna and 5 ul of the master mix.
the primers for the pcr reaction were designed using mit bioinformatics package primer3 version 4.0.0 [ 31 ] and optimized using gradient pcr amplifications and real-time pcr validation of each primer pair against various stool samples from zanzibar, haiti, and africa. real-time pcr reactions were carried out in separate, 96-well optical pcr plates using a rotor-gene 6000 real-time pcr machine (corbett life science, sydney, nsw, australia). a 10 ul reaction comprised of 5 ul of dna and 5 ul of the master mix. the 10 ul master mix contained: 0.5 ul of eva green dye; 0.75 ul of 10x primer/probe mix; 0.15 ul of rox; and 0.25 ul of forward and reverse primers (5 um). cycling was carried out at a ramp rate of 42.5c for 10 minutes and then heated to 95c for 10 minutes. cycling was then performed at a ramp rate of 1.6c for 60 seconds, followed by 55c for 1 minute, with cycles repeated for 40 times. the results were analyzed with the rotor-gene 6000 software. standard curves were created using serial dilutions of the plasmid with the nuclear expression vector pcdna3.1, and used to construct the pcr efficiency (e) and correlation coefficient (r 2 ). s. japonicum positive and negative control samples were supplied by the national veterinary services laboratory, usa and the national museum of the united states, respectively, and positive and negative s. mansoni and s. haematobium control samples were also provided by the filariasis research reagent resource center, university of georgia, athens, ga. all standards and samples were tested in duplicate. the qpcr assay for s. mansoni designed in this study was compared with a published reference assay described by marcus et al. and cestari et al. [ 17, 30 ]. briefly, an s. mansoni positive and negative control sample were provided by the filariasis research reagent resource center and a sample from a gambian patient was provided by dr. david lejon, geneva, switzerland. additional control samples from zanzibar (s. mansoni), culex pipiens, and anopheles stephensi were kindly provided by dr. john werre, armauer hansen research institute and dr. paolo montalto, istituto superiore di sanità, rome, italy. samples from the three mosquito species were extracted with the phenol-chloroform method. the protocol used for mosquito extraction followed the manufacturer protocol from the qiagen dneasy blood & tissue kit (qiagen, germantown, md). extractions were performed in duplicate for each sample. for each extraction, a negative control was included in each run to confirm the absence of contamination. the qpcr was performed in 96-well optical pcr plates, in triplicate, using the primer/probe assays developed in this study and the published assays. amplification was performed in separate, 96-well optical pcr plates using a rotor-gene 6000 real-time pcr machine (corbett life science, sydney, nsw, australia). a 10 ul reaction comprised of 5 ul of dna and 5 ul of the master mix. 5ec8ef588b
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