scholarly journals Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

2021 ◽  
Author(s):  
Nikoletta Naoumi ◽  
Kleita Michaelidou ◽  
George Papadakis ◽  
Agapi E. Simaiaki ◽  
Roman Fernandez ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Liquid Biopsy ◽  
Point Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Braf V600e ◽  
Analytical Sensitivity ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next-generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

scholarly journals Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR

Oncotarget ◽  
2017 ◽  
Vol 8 (49) ◽  
pp. 86253-86263 ◽  
Author(s):  
Vera Kloten ◽  
Nadine Rüchel ◽  
Nadina Ortiz Brüchle ◽  
Janina Gasthaus ◽  
Nils Freudenmacher ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Dna Extraction ◽  
Liquid Biopsy ◽  
Absolute Quantification ◽  
Circulating Dna ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Clinical utility of a liquid biopsy diagnostic approach in lung cancer using amplicon-based next-generation sequencing in parallel with allele-specific PCR.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e21516-e21516
Author(s):  
Yukti Choudhury ◽  
Wai Min Phyo ◽  
Jing Shan Lim ◽  
Chaitanya Gupta ◽  
Yiliang Ho ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Liquid Biopsy ◽  
Diagnostic Yield ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Advanced Lung Cancer ◽  
Parallel Testing ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

e21516 Background: The utility of a liquid biopsy test for diagnosis and treatment is determined by its accuracy, molecular target coverage and timeliness to a clinically informative result. We describe our clinical experience of providing comprehensive next-generation sequencing (NGS) plasma testing for advanced lung cancer cases, including parallel testing with allele-specific PCR (AS-PCR) for rapid EGFR mutation detection. Methods: Plasma cell-free DNA (cfDNA) from advanced lung cancer patients (n = 374) underwent real-world testing with an amplicon-based NGS assay, in a CAP and ISO15189 accredited central laboratory. The assay covers 51 genes, including 8 guideline recommended biomarkers- EGFR, BRAF, MET, ALK, RET, ROS1, ERBB2 and KRAS. 168 cases (44.9%) were treatment-naïve (baseline) and 206 cases (55.1%) had received one or more lines of treatment. Parallel testing with AS-PCR for 10 specific EGFR mutations was done for 151 cases (90 baseline, 61 non-baseline). Concordance of EGFR mutation detection by the two methods, and the frequency of detection of clinically actionable (driver) mutations by NGS were assessed. Turnaround time (TAT) was calculated from sample receipt. Results: An overall concordance of 97.4% was observed for EGFR mutations between AS-PCR and NGS. When restricted to baseline cases, concordance was 100%. Among baseline cases which were concordant with AS-PCR for EGFR negativity (n = 63), driver mutations were identified in EGFR (n = 7 rarer mutations), KRAS, ERBB2, MET, NRAS, BRAF and ALK (n = 16 total) by NGS, providing an additional 37% diagnostic yield (23/63 cases). Among all baseline cases tested by NGS, a driver gene mutation was found in 64.28% of cases, including EGFR (36.9%) and KRAS mutations (11.9 %). Among non-baseline cases which were EGFR-negative by NGS and by AS-PCR, added diagnostic yield provided by NGS was 33.3% (10 of 30 cases), and included 16.7% rarer EGFR mutations. Overall, detection rate of ALK, RET, ROS1 fusions was 2.4% (n = 5, 3 and 1, respectively). Median TAT for EGFR results by AS-PCR was 1 day (range 1-2 days), while median TAT for NGS results was 10 days (range 4-13 days). Conclusions: We report excellent real-world performance of blood-based liquid biopsy testing for detecting recommended biomarkers in lung cancers, including an approach combining two orthogonal platforms for quick decision making. Clinically meaningful diagnostic yields can be obtained using a timely comprehensive NGS assay, either individually or in parallel with rapid AS-PCR testing.

scholarly journals A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation

2013 ◽  
Vol 15 (2) ◽  
pp. 248-254 ◽  
Author(s):  
Philippe Szankasi ◽  
N. Scott Reading ◽  
Cecily P. Vaughn ◽  
Josef T. Prchal ◽  
David W. Bahler ◽  
...  
Keyword(s):  
Braf V600e Mutation ◽  
Braf V600e ◽  
Specific Pcr ◽  
Control Plasmid ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Pcr Test

scholarly journals Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

2017 ◽  
Author(s):  
Sergey V. Prykhozhij ◽  
Charlotte Fuller ◽  
Shelby L. Steele ◽  
Chansey J. Veinotte ◽  
Babak Razaghi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Point Mutations ◽  
Mutation Site ◽  
Transmission Rates ◽  
Specific Pcr ◽  
Allele Specific ◽  
Pcr Assays ◽  
Combination Of Methods ◽  
Mutation Strategies ◽  
Allele Specific Pcr

AbstractSingle-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

scholarly journals Improved detection of BRAF V600E using allele-specific PCR coupled with external and internal controllers

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Zhao Yang ◽  
Na Zhao ◽  
Dong Chen ◽  
Kun Wei ◽  
Ning Su ◽  
...  
Keyword(s):  
Braf V600e ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Helicobacter pylori 23S rRNA gene A2142G, A2143G, T2182C, and C2195T mutations associated with clarithromycin resistance detected in Sudanese patients

2020 ◽  
Author(s):  
Aalaa Mahgoub Albasha ◽  
Maram M. Alnosh ◽  
Esraa Hassan Osman ◽  
Duha M Zeinalabdin ◽  
Amira A M Fadl ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene ◽  
Allele Specific ◽  
H Pylori ◽  
Allele Specific Pcr

Abstract Background: Clarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms.Materials and Methods: Two hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primers targeting 16S rRNA and 23S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations.Results: Out of 288 samples, H. pylori was detected in 97 (33.7%) sample. Allele-specific PCR detected the variant A2142G in 9/97 (9.3%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 48% (12/25) of samples; the A2142G was present in one sample, A2143G in 5 samples, T2182C in 4 samples, and C2195T in 3 samples. There was no association of 23S rRNA gene point mutations with gender, age group and geographical distribution of patients.Conclusion: This study revealed a high frequency (48%) of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene's V domain. This information should be taken into consideration before choosing optimal therapy for H. pylori eradication.

scholarly journals Rapid Detection of Point Mutations Conferring Resistance to Fluoroquinolone in gyrA of Helicobacter pylori by Allele-Specific PCR

2006 ◽  
Vol 45 (2) ◽  
pp. 303-305 ◽  
Author(s):  
T. Nishizawa ◽  
H. Suzuki ◽  
A. Umezawa ◽  
H. Muraoka ◽  
E. Iwasaki ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Rapid Detection ◽  
Point Mutations ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Helicobacter pylori 23S rRNA gene A2142G, A2143G, T2182C, and C2195T mutations associated with clarithromycin resistance detected in Sudanese patients

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Aalaa Mahgoub Albasha ◽  
Maram M. Elnosh ◽  
Esraa Hassan Osman ◽  
Duha M. Zeinalabdin ◽  
Amira A. M. Fadl ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene ◽  
Allele Specific ◽  
H Pylori ◽  
Allele Specific Pcr

Abstract Background Clarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms. Materials and methods Two hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primer targeting 16S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations. Results Out of 288 samples, H. pylori was detected in 88 (~ 30.6%) samples by 16 s RNA. Allele-specific PCR detected the variant A2142G in 9/53 (~ 17%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 36% (9/25) of samples; the A2142G was present in one sample, A2143G in 5 samples and T2182C in 4 samples. Additionally, another point mutation (C2195T) was detected in 3 samples. There was no association of 23S rRNA gene point mutations with gender, age group, and patients’ geographical distribution. Conclusion This study revealed a high frequency (36%) of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene’s V domain. This information should be taken into consideration to avoid eradication therapy failing.

scholarly journals Mutations Affecting the Rossman Fold of Isoleucyl-tRNA Synthetase Are Correlated with Low-Level Mupirocin Resistance in Staphylococcus aureus

2002 ◽  
Vol 46 (2) ◽  
pp. 438-442 ◽  
Author(s):  
Martin Antonio ◽  
Neil McFerran ◽  
Mark J. Pallen
Keyword(s):  
Staphylococcus Aureus ◽  
Point Mutations ◽  
Trna Synthetase ◽  
Low Level ◽  
Specific Pcr ◽  
Uncertain Significance ◽  
Allele Specific ◽  
Mupirocin Resistance ◽  
Allele Specific Pcr

ABSTRACT The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 μg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.

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