scholarly journals Development and Application of a Tri-allelic PCR Assay for ScreeningVgsc-L1014FKdrMutations Associated with Pyrethroid and Organochlorine Resistance in the MosquitoCulex quinquefasciatus

2019 ◽  
Author(s):  
Walter Fabricio Silva Martins ◽  
Bárbara Natieli Silva Pereira ◽  
Ana Thayse Vieira Alves ◽  
Annabel Murphy ◽  
Paulo Geovani Silva Martins ◽  
...  
Keyword(s):  
Allelic Variation ◽  
High Specificity ◽  
Cost Effective ◽  
Diagnostic Assay ◽  
Pcr Assay ◽  
Public Health Importance ◽  
Tropical Regions ◽  
Vector Borne Diseases ◽  
Specific Pcr ◽  
Allele Specific

AbstractBackgroundCulex quinquefasciatus,has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. InC. quinquefasciatustwo non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of theVgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda.ResultsThis new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches – pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles atVgsc-L1014F, with genotyping results strongly correlated with 98.64% and 100% against pyrosequencing and TaqMan, respectively.ConclusionsOur results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag ® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9 , HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene

2018 ◽  
Vol 5 (2) ◽  
pp. e92
Author(s):  
Anuj Kumar Tyagi ◽  
Mary Boudal Khoshbeen ◽  
Patricia Huezo-Diaz Curtis ◽  
Chakradhara Rao S. Uppugunduri ◽  
Marc Ansari
Keyword(s):  
Dna Methyltransferase ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Pcr Assay ◽  
Mgmt Gene ◽  
Methylguanine Dna Methyltransferase ◽  
Specific Pcr ◽  
Allele Specific ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression.These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing,   validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments.Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant. 

scholarly journals Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies

2021 ◽  
Author(s):  
TINA BAGRATUNI ◽  
Athina N Markou ◽  
Dimitrios Patseas ◽  
Nefeli Mavrianou-Koutsoukou ◽  
Foteini Aktypi ◽  
...  
Keyword(s):  
Cost Effective ◽  
Pcr Assay ◽  
Cell Free Dna ◽  
Monoclonal Gammopathies ◽  
Specific Pcr ◽  
Free Dna ◽  
Highly Sensitive ◽  
Sequential Samples ◽  
Tumor Load ◽  
Allele Specific

We here describe a novel method for the detection of MYD88L265P mutation using a competitive allele specific PCR (Cast-PCR) assay. This assay has a sensitivity of 1x10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg) and allowed the detection of MYD88L265P somatic mutation in both tumor derived DNA (tDNA) and cell free DNA (cfDNA). In addition, using Cast-PCR assay we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with IgM-MGUS and 110 with WM, of which 54 were asymptomatic and 56 symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared to asymptomatic WM and in those with asymptomatic WM compared to IgM-MGUS patients. In addition, the evaluation of sequential samples showed that MAF decreased after treatment while increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly-sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tumor and cell free DNA samples which also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

scholarly journals Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Cai-Ying Zhu ◽  
Chun-Chun Zhao ◽  
Yi-Guan Wang ◽  
De-Ling Ma ◽  
Xiu-Ping Song ◽  
...  
Keyword(s):  
Aedes Albopictus ◽  
Large Scale ◽  
Resistance Mechanism ◽  
High Specificity ◽  
Cost Effective ◽  
Knockdown Resistance ◽  
Dengue Vector ◽  
Time Saving ◽  
Specific Pcr ◽  
Allele Specific

Abstract Background Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-saving and cost-effective procedure to monitor the alleles in large-scale studies. Methods Based on the mutation genotypes of the 1534 locus in the kdr gene, F/F, F/S, F/C, F/L, S/S, C/C, L/L and S/C, we designed specific forward and reverse primers and optimized the reaction conditions for establishing of the allele-specific PCR(AS-PCR) detection technique. DNA sequencing in this study was taken as the gold standard, and used to determine the accuracy of AS-PCR. Results The designed AS-PCR technique showed high specificity for distinguishing the mutations at the 1534 locus, as the accuracy for F/F, F/S, F/C, F/L, S/S, C/C and S/C were 100%, 95.35%, 100%, 100%, 100%, 100% and 100%, respectively. Conclusions The designed AS-PCR technique effectively distinguished individual genotypes for the mutations at the 1534 locus in the kdr gene, which could facilitate the knockdown resistance surveillance in Ae. albopictus in large-scale studies.

scholarly journals A novel ultra-sensitive urine based FGFR3 mutation assay for diagnosis and recurrence detection of non-muscle-invasive bladder cancer (NMIBC) – Design of the Urodiag® Kit

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Fgfr3 Mutation ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required the amplification steps and the PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild type). In the development of Urodiag ® Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine.Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays.Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

BMC Genomics ◽  
2007 ◽  
Vol 8 (1) ◽  
pp. 275 ◽  
Author(s):  
Pongsakorn Wangkumhang ◽  
Kridsadakorn Chaichoompu ◽  
Chumpol Ngamphiw ◽  
Uttapong Ruangrit ◽  
Juntima Chanprasert ◽  
...  
Keyword(s):  
Pcr Assay ◽  
Web Based ◽  
Specific Pcr ◽  
Allele Specific ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

Novel Assay for the Identification of NOTCH1 PEST Domain Mutations Using Fragment Analysis and Allele Specific PCR

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5127-5127
Author(s):  
Paulo Vidal Campregher ◽  
Roberta Cardoso Petroni ◽  
Nair Muto ◽  
Rubia Santana ◽  
Roberta Sitnik ◽  
...  
Keyword(s):  
Cost Effective ◽  
Single Mutation ◽  
Wild Type ◽  
Double Peak ◽  
Fragment Analysis ◽  
Specific Pcr ◽  
Routine Identification ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Notch1 Mutations

Abstract Abstract 5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c. 7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34. We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c. 7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs: A single 391 bp peak: wild type samplesThree peaks (391 bp, 389 bp and 356 bp): heterozygous for c. 7544_7545delCTTwo peaks (391 bp and another bigger or smaller, depending on the size of insertion/deletion): another insertion or deletion, but not c. 7544_7545delCT. We have studied 46 de-identified blood samples from patients with CLL, in several diverse stages, using our assay. In 40 patients, there was no NOTCH1 mutation detected. Six patients had a pattern compatible with c. 7544_7545delCT NOTCH1 mutation (see figure 1), and no patients presented with another mutation. Overall the frequency of NOTCH1 mutations in our series was 13 %. Selected mutated samples were confirmed through amplicon sequencing. In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations using fragment analysis and allele specific pcr that is suitable for implementation in the clinical setting for CLL patients evaluation. We will continue testing more CLL patients in order to identify another, rarer, NOTCH1 mutations. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations . / A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. . / B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Disclosures: No relevant conflicts of interest to declare.

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