scholarly journals Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Sollip Kim ◽  
Hye Won Lee ◽  
Woochang Lee ◽  
Sail Chun ◽  
Won-Ki Min
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Sequencing ◽  
Reference Method ◽  
Korean Population ◽  
Clinical Samples ◽  
Tpmt Activity ◽  
Bone Marrow Toxicity ◽  
Thiopurine Methyltransferase ◽  
Allele Specific

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered.TPMTgenotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population.Materials and Methods. Three majorTPMTsingle-nucleotide polymorphisms (TPMT*2, *3B, and *3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing.Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency ofTPMT*3Cwas 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. NoTPMT*2, *3A, or *3Balleles were observed in this Korean population.Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

scholarly journals Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

2011 ◽  
Vol 49 (9) ◽  
pp. 3168-3174 ◽  
Author(s):  
Andrew S. Bae ◽  
Karin S. Ku ◽  
Michael D. Miller ◽  
Hongmei Mo ◽  
Evguenia S. Svarovskaia
Keyword(s):  
Hepatitis C ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Genotype 1A ◽  
Allele Specific

scholarly journals Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

2015 ◽  
Vol 53 (3) ◽  
pp. 930-940 ◽  
Author(s):  
Iker A. Sevilla ◽  
Elena Molina ◽  
Natalia Elguezabal ◽  
Valentín Pérez ◽  
Joseba M. Garrido ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Reference Method ◽  
Clinical Samples ◽  
Dna Amount ◽  
Content Type ◽  
Specificity And Sensitivity

Mycobacterium tuberculosiscomplex,Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection ofMycobacteriumgenus,M. aviumsubspecies, andM. tuberculosiscomplex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n= 38) and nonmycobacterial (n= 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n= 57), archival clinical samples (n= 233), and strains isolated from various hosts (n= 147). The minimum detectable DNA amount per reaction was 50 fg forM. bovisBCG andM. kansasiiand 5 fg forM. aviumsubsp.hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identifyM. aviumandM. tuberculosiscomplex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

scholarly journals Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR

2004 ◽  
Vol 50 (2) ◽  
pp. 306-312 ◽  
Author(s):  
Stefan S Biel ◽  
Andreas Nitsche ◽  
Andreas Kurth ◽  
Wolfgang Siegert ◽  
Muhsin Özel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow ◽  
Real Time ◽  
Bone Marrow Transplant ◽  
Real Time Pcr ◽  
Marrow Transplant ◽  
Test System ◽  
Urine Samples ◽  
Clinical Samples ◽  
Transplant Patients

Abstract Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 106 genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <103 GE/mL and 100% for urine samples containing 109 GE/mL. Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

2013 ◽  
Vol 44 (2) ◽  
pp. 505-510 ◽  
Author(s):  
Aline Padilha Fraga ◽  
Tatiana de Vargas ◽  
Nilo Ikuta ◽  
André Salvador Kazantzi Fonseca ◽  
Álvaro José Celmer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Gallisepticum ◽  
Clinical Samples ◽  
Mycoplasma Synoviae ◽  
Commercial Poultry

scholarly journals Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

2020 ◽  
Author(s):  
Vu Thuy Duong ◽  
Le Thi Phuong Tu ◽  
Ha Thanh Tuyen ◽  
Le Thi Quynh Nhi ◽  
James I Campbell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Middle Income ◽  
Middle Income Countries ◽  
Virulence Markers ◽  
Low Middle Income Countries ◽  
High Prevalence ◽  
Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

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