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    Direct loop-mediated isothermal amplification from Plasmodium chabaudi infected blood samples:
    inability to discriminate genomic and cDNA sequences (2012)

    Art
    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Autoren
    Ebbinghaus, Petra
    von Samson-Himmelstjerna, Georg (WE 13)
    Krücken, Jürgen (WE 13)
    Quelle
    Experimental parasitology; 131(1) — S. 40–44
    ISSN: 1090-2449
    Sprache
    Englisch
    Verweise
    DOI: 10.1016/j.exppara.2012.02.024
    Pubmed: 22417971
    Kontakt
    Institut für Parasitologie und Tropenveterinärmedizin

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35, 22, 23
    14163 Berlin
    +49 30 838 62310
    parasitologie@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    Loop-mediated isothermal amplification (LAMP) has been increasingly used for diagnosis and quantification of pathogens. Since the Bst DNA polymerase used in this assay is highly resistant to PCR inhibitors present in blood, direct analysis of blood samples without DNA or RNA extraction is possible. Indeed, the presence of Plasmodium chabaudi specific nucleic acids was easily detectable using primer sets for P. chabaudi 18S rRNA and the cir 1 mRNA. Despite the fact that primers for cir 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin II mRNAs were used that spanned an intron, selective amplification of mRNA in the presence of contaminating genomic DNA was not possible. Optimization of the reaction temperature could only improve discrimination when low complexity templates (target sequences cloned in a plasmid vector) were used. Placing different LAMP primers across intron exon boundaries did not prevent amplification in the absence of reverse transcriptase. Probably due to the high A+T content and low number of introns only a very limited number of possible primer sets spanning introns could be identified in the target genes and no reaction conditions could be established that would allow quantification of RNA levels in the presence of DNA directly from blood samples.