Evaluation of droplet digital PCR and next generation sequencing for characterizing DNA reference material for KRAS mutation detection.
Sci Rep
; 8(1): 9650, 2018 11 30.
Article
en En
| MEDLINE
| ID: mdl-30504843
ABSTRACT
KRAS gene mutations are predictive markers of non-response to anti-epidermal growth factor receptor. An increasing number of techniques are being developed to detect KRAS mutations. To obtain consistent and comparable results, a traceable reference material (RM) is necessary for validation the routinely used method. However, a lack of reference methods is a main impediment for deriving traceability and measurement comparability. In this study, droplet digital PCR (ddPCR) and next generation sequencing (NGS) were evaluated. No cross- reactivity was detected with any of the probe by ddPCR. The measured fraction of KRAS mutant allele by ddPCR and NGS agreed with the prepared value by gravimetrical dilution (concordance (k) >0.95 and >0.93 for ddPCR and NGS, respectively). The reliable limit of quantification (LOQ) was 0.1% and 1% for ddPCR and NGS, respectively. In conclusion, the validated ddPCR and NGS are suitable to characterize the KRAS RM due to the high specificity and accuracy. Verification of the LOD of three commercial kits by using the NIM-KRAS-8 RM showed that the LOD was inconsistent with the claimed LOD of the kits (1%) for some assays. This indicates a traceable RM was important for setting up the criteria regarding the LOD for the commercial kit.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa
/
Proteínas Proto-Oncogénicas p21(ras)
/
Análisis de Secuencia de ADN
/
Secuenciación de Nucleótidos de Alto Rendimiento
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2018
Tipo del documento:
Article