Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA
Mem. Inst. Oswaldo Cruz
; 101(supl.1): 133-136, Oct. 2006. graf
Article
in English
| LILACS
| ID: lil-441237
Responsible library:
BR1.1
ABSTRACT
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 (R²) 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°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.
Full text:
Available
Collection:
International databases
Health context:
Neglected Diseases
Health problem:
Helminthiasis
/
Neglected Diseases
/
Schistosomiasis
/
Zoonoses
Database:
LILACS
Main subject:
Schistosoma mansoni
/
Schistosomiasis mansoni
/
Polymerase Chain Reaction
/
DNA, Helminth
Type of study:
Diagnostic study
/
Practice guideline
Limits:
Animals
/
Humans
Language:
English
Journal:
Mem. Inst. Oswaldo Cruz
Journal subject:
Tropical Medicine
/
Parasitology
Year:
2006
Document type:
Article
Affiliation country:
Brazil
Institution/Affiliation country:
Fiocruz/BR