Standardization of a two-step real-time polymerase chain reaction based method for species-specific detection of medically important Aspergillus species.
Indian J Med Microbiol
; 35(3): 381-388, 2017.
Article
em En
| MEDLINE
| ID: mdl-29063883
ABSTRACT
PURPOSE:
Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. MATERIALS ANDMETHODS:
Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger).RESULTS:
Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml.CONCLUSION:
The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Aspergilose
/
Aspergillus
/
Técnicas de Diagnóstico Molecular
/
Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
/
Guideline
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Indian J Med Microbiol
Assunto da revista:
MICROBIOLOGIA
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Índia