Rapid HLA-B27 screening with real-time TaqMan PCR: a clinical validation in the Dutch population

2011 ◽  
Vol 49 (12) ◽  
Author(s):  
Elianne A. Roelandse-Koop ◽  
Bert Buisman ◽  
Erik J. van Hannen ◽  
Anneke van der Zee ◽  
Wouter Kortlandt ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Human Leukocyte ◽  
Clinical Samples ◽  
Correct Result ◽  
Hla B27 ◽  
Dutch Population ◽  
Exon 2 ◽  
Allele Specific ◽  
Taqman Pcr

AbstractHuman leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis.We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control.A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.

scholarly journals TaqMan-Based Real-Time PCR for Genotyping Common Polymorphisms of Haptoglobin (HP1 and HP2)

2008 ◽  
Vol 54 (11) ◽  
pp. 1908-1913 ◽  
Author(s):  
Mikiko Soejima ◽  
Yoshiro Koda
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Relative Number ◽  
High Throughput Analysis ◽  
Large Populations ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Pcr Method ◽  
The Difference

Abstract Background: The haptoglobin gene (HP) has 2 common codominant alleles (HP1 and HP2) that account for 3 phenotypes. HP2 is generated by a 1.7-kb intragenic duplication of HP1. Methods: We used the real-time TaqMan PCR system to develop an effective method for HP genotyping that allows us to evaluate the relative number of copies of the HP2 allele–specific junctional region of the 1.7-kb gene duplication (HP2) by comparing the intensity of the amplification signals to those of the HP promoter region (HP5′), which was used as the internal control. The difference in threshold cycles (ΔCt) between HP2 and HP5′ was used to assess HP2 copy number. In addition, the assay detects the HP deletion (HPdel) at the same time. Results: The mean 2−ΔΔCt values (the HP2/HP5′ ratio) obtained from 123 samples of known HP genotypes clearly differentiated 2 nonoverlapping intervals that correspond to the HP genotypes. Ratios for HP2/HP1 samples ranged from 0.34–0.50, HP2/HP2 samples ranged from 0.79–0.98, and the absence of an HP2 allele signal was defined as HP1/HP1. We simultaneously detected HPdel. The assay produces results in <1 h. Conclusions: The TaqMan-based real-time PCR method was successfully applied to HP genotyping. The method is easy to use in a molecular diagnosis laboratory, and its robustness and rapidity make it suitable for high-throughput analysis of large populations.

scholarly journals STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms as markers of predisposition to juvenile idiopathic arthritis. What can genetics give to understand its heterogeneity?

2019 ◽  
Vol 13 (4) ◽  
pp. 55-60
Author(s):  
E. S. Fedorov ◽  
M. Yu. Krylov ◽  
S. O. Salugina ◽  
E. Yu. Samarkina ◽  
A. N. Latypova
Keyword(s):  
Juvenile Idiopathic Arthritis ◽  
High Risk ◽  
Antinuclear Antibody ◽  
Pediatric Population ◽  
Human Leukocyte ◽  
Control Group ◽  
Entire Group ◽  
Hla B27 ◽  
Systemic Jia ◽  
Allele Specific

Juvenile idiopathic arthritis (JIA) is a multifactorial immune-mediated inflammatory disease in childhood, the most common type of rheumatic disease in children. It is characterized by the polygenic type of hereditary predisposition.Objective: to study the association of STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms with the predisposition to certain JIA subtypes in the Russian pediatric population.Patients and methods. The investigation enrolled 177 patients, including 66 patients diagnosed with JIA and 111 healthy unrelated volunteers (a control group). Of the 66 patients with JIA there were 30 (45%) with oligoarthritis: 20 (67%) with human leukocyte antigen B27(HLA-B27)-positive JIA (that was associated with enthesitis, HLA-B27 positive JIA (JIA-B27), 10 (33%) with anterior uveitis concurrent with antinuclear antibody-positive JIA (JIA-uveitis); 20 (30%) with polyarticular JIA (JIA-poly), seronegative for rheumatoid factor; and 16 (24%) with systemic JIA (JIA-sys). As a control for genotyping STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms, the investigators studied 103 and 111 DNA samples from healthy adult volunteers, respectively. STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms were investigated using allele-specific real-time polymerase chain reaction (RT-PCR).Results and discussion. In the oligoarticular JIA group, the frequency of the STAT4 T allele was significantly higher than that in the control group (38.3 and 20.4%, respectively; p=0.004). This allele was also significantly more common in the JIA-B27 (35.0 and 20.4%, respectively; p=0.044) and JIA-uveitis (45.0 and 20.4%, respectively; p=0.021) groups compared with the control one. No significant differences were found in the frequency of the mutant STAT4 T allele between the control group and the JIA-sys and JIA-poly groups. Regression analysis showed that the identification of the STAT4 T allele was associated with the high risk of a predisposition to oligoarticular JIA as a whole (odds ratio, OR 2.43; 95% confidence interval (CI) 1.23–4.70; p=0.007), as well as to the antinuclear antibody-positive oligoarticular JIA with uveitis (JIA-uveitis): the risk in T allele carriers was 3.2 times higher than that in the control (OR 3.19; 95% CI 1.09–9.06; p= ). A high risk for predisposition was also found in the JIA-B27 subgroup compared with the control (OR 2.10; 95% CI 0.38–4.60; p=0.070). There were no statistical differences in the frequency of genotypes and alleles of the IRF5 rs2004640 G/T polymorphism between the entire group of JIA as a whole and its individual clinical types, as well as the control group.Conclusion. This pilot study confirmed that the STAT4 rs7574865 G/T polymorphism was associated with the risk of oligoarticular JIA, mainly that of JIA-uveitis and JIA-B27.

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

2005 ◽  
Vol 76 (3) ◽  
pp. 350-355 ◽  
Author(s):  
Lawrence Corey ◽  
Meei-Li Huang ◽  
Stacy Selke ◽  
Anna Wald
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Taqman Pcr ◽  
Simplex Virus

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 Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 835-840 ◽  
Author(s):  
S Simsek ◽  
NM Faber ◽  
PM Bleeker ◽  
AB Vlekke ◽  
E Huiskes ◽  
...  
Keyword(s):  
Internal Control ◽  
Human Platelet ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Dutch Population ◽  
Reliable Technique ◽  
Platelet Antigen ◽  
Allele Specific ◽  
Specific Restriction

Abstract Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an “internal control” for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.

scholarly journals Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Cristin Roma ◽  
Claudia Esposito ◽  
Anna Maria Rachiglio ◽  
Raffaella Pasquale ◽  
Alessia Iannaccone ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Response To Treatment ◽  
Egfr Mutations ◽  
Clinical Samples ◽  
Training Set ◽  
Similar Frequency ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Validation Set

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct=37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

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 Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

2022 ◽  
Vol 9 (1) ◽  
pp. 1-20
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

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