Dataset Information

Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR.

ABSTRACT:

Unlabelled

Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ?1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies.

Conclusion

Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting.

SUBMITTER: Orue A

PROVIDER: S-EPMC5025196 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR.

Orue Andrea A Rieber Manuel M

PloS one 20160915 9

<h4>Unlabelled</h4>Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive sample ...[more]

PMID: 27632281

Similar Datasets

Project description:Epithelial growth factor receptor (EGFR)-tyrosine kinase inhibitors are the standard first-line treatment for patients with metastatic non-small cell lung cancer (NSCLC) expressing sensitive EGFR-mutants. Other drugs target different driver mutants, including the serine/threonine-protein kinase B-raf (BRAF) inhibitor dabrafenib, which has exhibited promising efficacy for treating patients with metastatic BRAF-mutated NSCLC. Therefore, identifying patients carrying mutations that may be treated using targeted therapies is important. However, the methods of molecular detection presently applied in clinical practice, particularly detection of BRAF in NSCLC patients, require further investigation. Therefore, more sensitive and economic methods are required. The present study applied the competitive allele-specific TaqMan polymerase chain reaction (CastPCR) technology to the molecular detection of EGFR (del2235-2249, del2236-2250, T790M, L858R) and BRAF (V600E, G469A, D594G) mutations in 144 treatment-naive patients with lung adenocarcinoma, and analyzed the association between the mutation rates and patients' clinicopathological features. 51.4% (74/144) cases were identified harboring EGFR mutations. A total of 40.3% (58/144) patients carried sensitizing mutations (exon 19 deletion or L858R) and 14.6% (21/144) carried T790M mutations. 6.9% (10/144) mutation-positive patients were double-mutated. Total EGFR mutation rate was significantly increased in female compared with that of males (60.9 vs. 43.8%, P<0.05), in non-smokers compared with that of smokers (62.8 vs. 34.5%, P<0.05). In total, 8.3% (12/144) patients were identified with BRAF mutations. 16.7% were V600E (2/12) and 83.3% (10/12) were non-V600E mutants. Among the 10 non-V600E mutations, D594G accounted for 90.0% (9/10) and G469A accounted for 10.0% (1/10). Statistical analysis demonstrated that the BRAF mutation rate was not associated with any of the following clinicopathological features: Sex, age, smoking history, clinical stages, distant metastasis, differentiation degree, tumor size and regional lymph node metastasis (P≥0.05). CastPCR technology is a robust method with high sensitivity for the molecular detection of EGFR and BRAF mutations in clinical formalin-fixed paraffin-embedded samples.

Project description:BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.

Project description:Kefir grains are complex microbial systems of several groups of microorganisms. The identification and quantification of the microbial composition of milk kefirs was described in several studies, which provided an insight into the microbial consortia in this complex ecosystem. Nevertheless, the current methods for identification and quantification are not appropriate for deeper studies on kefir consortia, e.g., population dynamics and microbial interactions in kefir grains. This requires another sensitive and reliable quantitative method. Therefore, this study aims to develop multiplexed qPCR assays to specifically detect and quantify, as an example, several microorganisms of the milk kefir microbial community. Primer-probe sets, which target species-specific genes in six bacteria and five yeasts, were designed, and their sensitivity and specificity to the target species was analyzed in simplex as well as four multiplex qPCR assays. The self-designed multiplex assays were applied for the detection of target bacteria and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all target microorganisms in simplex and multiplex qPCR was achieved by good linearity, efficiency, repeatability and reproducibility in all assays. When the designed assays were applied on six kefirs, all target microorganisms were detected in different samples, but not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens and Lb. kefiri were present in all six kefirs studied, but were associated with different other yeasts and bacteria. Especially on the yeast community a significant diversity was observed. In general, multiplex TaqMan qPCR as developed here was proven to have high potential for specific identification of target microorganisms in kefir samples and for the first time, eleven target bacteria and yeasts of kefir microbiota were rapidly detected and quantified. This study, thus, provides a fast and reliable protocol for future studies on kefir and other similar microbial ecosystems.

Dataset Information

Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR.

Unlabelled

Conclusion

Publications

Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets