Direct loop-mediated isothermal amplification from Plasmodium chabaudi infected blood samples: inability to discriminate genomic and cDNA sequences.
Exp Parasitol
; 131(1): 40-4, 2012 May.
Article
en En
| MEDLINE
| ID: mdl-22417971
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN Protozoario
/
Plasmodium chabaudi
/
ADN Complementario
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Técnicas de Amplificación de Ácido Nucleico
/
Malaria
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Exp Parasitol
Año:
2012
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Estados Unidos