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 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).

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.

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.

Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine.

2011 ◽  
Vol 29 (7_suppl) ◽  
pp. 271-271
Author(s):  
J. Millholland ◽  
S. G. Patel ◽  
C. A. Fernandez ◽  
A. P. Shuber
Keyword(s):  
Bladder Cancer ◽  
Real Time ◽  
Mutation Analysis ◽  
Real Time Pcr ◽  
Mutation Detection ◽  
Cost Effective ◽  
Optimal Performance ◽  
Single Mutation ◽  
Pcr Assay ◽  
Fgfr3 Mutations

271 Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary matrix metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. The LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. Results: To validate the new multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. [Table: see text]

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.

Clonality Assay (X-CIP) and JAK 2 V617P Mutation: Clustering Patients with Essential Thrombocythemia at High Risk for Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2597-2597
Author(s):  
Alessia Fiorini ◽  
Giovanni Reddiconto ◽  
Giuliana Farina ◽  
Patrizia Chiusolo ◽  
Luciana Teofili ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
High Risk ◽  
Thrombotic Event ◽  
Young Female ◽  
Wild Type ◽  
Jak2 Mutation ◽  
Specific Pcr ◽  
Allele Specific ◽  
Risk For Thrombosis ◽  
Allele Specific Pcr

Abstract We have previously demonstrated that clonal hemopoiesis in female ET patients was significantly associated to the risk of thrombosis. When data on JAK2 V617P mutation became available we aimed to evaluate the incidence of this mutation according to clonality status. Ninety-two female patients, median age 32 (19–65) with a diagnosis of ET according to PVSG criteria were studied for clonality with X-CIP, 83 patients were evaluable for endogenous erythroid colonies (EECs) growth and 44 patients were evaluable for JAK2 V617P mutation. Twenty episodes of thromboses (21.7%) were detected and they include splanchnic (11), CNS (5), limb (3) thromboses and pulmonary embolism (1). Thromboses were mainly detected at diagnosis. Thirthy-seven patients showed clonal hemopoiesis (40%), 27 had polyclonal hemopoiesis (29.5%) and 28 were considered uninterpretable due to constitutional skewing (30.5%). Thromboses were overrepresented in the monoclonal group in respect to the polyclonal one (15/37 vs 2/27, p=0.003). DNA for analysis of JAK2 V617P by allele-specific PCR was available for 44 of these patients. Thirty-two patients of 44 (73%) showed JAK2 V617P mutation; JAK2 V617P was found in 16/21 patients with monoclonal hemopoiesis (76%), in 9/13 in the polyclonal group (69%) and in 7/10 (70%) in patients with constitutional skewing. Thirteen of the 32 patients with JAK2 V617P mutation (41%) had thromboses while no thrombotic event was recorded in wild type patients (p=0.008); among the 13 patients with thrombosis and JAK2 V617P mutation, 9 (69%) had monoclonal hemopoiesis, 2 (15%) had polyclonal hemopoiesis, and 2 had constitutive skewing. Thus there was a significant increase in thrombotic events in patients with JAK2 V617P mutation and monoclonal hemopoiesis (p=0.04). JAK2 V617P mutation was also significantly associated to the presence of EECs (p=0.02); in fact 22/26 patients with EECs growth showed JAK2 mutation (85%). Finally when patients with splanchnic thromboses were analyzed 9/11 had a monoclonal X-CIP (81.8%) and 7 of them who were evaluable for JAK2 V617P mutation were invariably carriers of the mutation. Thus we confirm that X-CIP in young female ET is correlated to the risk of thrombosis. JAK2 V617P mutation is significantly associated to the development of thrombosis and is present in the vast majority of patients with monoclonal hemopoiesis. The mechanisms underlying monoclonal hemopoiesis in the absence of JAK2 V617P mutation are unclear, notwithstanding these patients are clinically characterized by both absence of EECs and thrombosis.

scholarly journals Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR

2015 ◽  
Vol 53 (11) ◽  
pp. 3418-3422 ◽  
Author(s):  
Zengguo Wang ◽  
Ruijun Han ◽  
Ying Liu ◽  
Quanli Du ◽  
Jifeng Liu ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Mutant Allele ◽  
Wild Type ◽  
Pcr Assay ◽  
Diagnostic Pcr ◽  
Content Type ◽  
Specific Pcr ◽  
Allele Specific ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

Resistance ofBordetella pertussisto erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistantB. pertussisdirectly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA ofB. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recentB. pertussisisolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistantB. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containingB. pertussisDNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetellabacterial pathogens and NP swab samples that did not contain the DNAs ofB. pertussiswere examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries.

Sign in / Sign up

Export Citation Format

Share Document