A primer and probe set for detecting multiple types of EGFR exon 19 deletions.
Anal Biochem
; 513: 61-67, 2016 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-27601284
ABSTRACT
EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sequência de Bases
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Sondas de DNA
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Derrame Pleural Maligno
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Deleção de Sequência
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Carcinoma Pulmonar de Células não Pequenas
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Ácidos Nucleicos Peptídicos
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Receptores ErbB
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Neoplasias Pulmonares
Limite:
Female
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Humans
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Male
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article