Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA.
This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R(2)) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83 degrees C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.
Gomes
Ana Lisa do Vale
AL
Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-Fiocruz, Av. Prof Moraes Rego s/n, 50670-420 Recife, PE, Brazil.
Melo
Fábio L
FL
Werkhauser
Roberto P
RP
Abath
Frederico G C
FG
D000818:Animals; D018509:DNA, Helminth / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012550:Schistosoma mansoni / Q000235:genetics*; D012555:Schistosomiasis mansoni / Q000175:diagnosis*; D012680:Sensitivity and Specificity
D018509:DNA, Helminth