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 Investigation on the deletion and duplication of PMP22 gene in patients with Charcot-Marie-Tooth using Real-time PCR in Chaharmahal and Bakhtiari and Isfahan Provinces

2019 ◽  
Vol 21 (6) ◽  
pp. 254-257
Author(s):  
Masoumeh Pourhadi ◽  
Morteza Hashemzadeh-Chaleshtori ◽  
Mostafa Gholami ◽  
Mohammad-Saeed Jami
Keyword(s):  
Gene Duplication ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pmp22 Gene ◽  
Charcot Marie Tooth ◽  
Qpcr Method ◽  
Pcr Method ◽  
Hereditary Patterns ◽  
Prevalent Form

Background and aims: Charcot-Marie-Tooth (CMT) is a common sensory-motor polyneuropathy with a prevalence of 1/2500. It is divided into different subgroups and has various hereditary patterns. Among the different subgroups of CMT, type 1A is the most prevalent form of the disease, which is created due to the duplication of the PMP22 gene. In patients has a deletion in the PMP22 gene, the hereditary neuropathic disease is known to be liable to pressure. The aim of this study was to identify the patients affected by the disease with the new, simple, and fast qPCR method and to investigate the appropriateness of this method in evaluating these types of mutations. Methods: In this analytical-descriptive study (code:IR.SKUMS.REC.1394.152), gene duplication and deletion in the patients were studied using the Excel software. The blood samples of 15 families afflicted with CMT and 49 healthy individuals were collected in EDTA anticoagulant tubes and analyzed. DNA extraction and quantitative real-time PCR method were applied for the PMP22 gene as the target gene and the albumin gene as the internal control gene. Results: Two genes were compared in each patient, and it was found that 46% of the subjects had duplication in the PMP22 gene. Conclusion: The qPCR method is an easy and fast way to detect gene duplication and deletion in CMT patients. It does not require any statistical software and can be performed without needing for parental DNA. In addition, the results of this study are consistent with the results of various studies in some countries of the world where the highest levels of deletion and duplication in PMP22 gene are seen.

scholarly journals SIMPLIFYING CELIAC DISEASE PREDISPOSING HLA-DQ ALLELES DETERMINATION BY THE REAL TIME PCR METHOD

2015 ◽  
Vol 52 (2) ◽  
pp. 143-146 ◽  
Author(s):  
Nicole SELLESKI ◽  
Lucas Malta ALMEIDA ◽  
Fernanda Coutinho de ALMEIDA ◽  
Lenora GANDOLFI ◽  
Riccardo PRATESI ◽  
...  
Keyword(s):  
Celiac Disease ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Specific Primers ◽  
The Real ◽  
Hla Dq ◽  
Pcr Method

Background Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value. Objective Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR) and melting curve analysis with the specificity of sequence-specific primers (SSP). Methods Amplifications of sequence-specific primers for DQA1*05 (DQ2), DQB1*02 (DQ2), and DQA1*03 (DQ8) were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results. Results Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%). One hundred fourteen samples (61%) were positive for a single allele, 68 (36.3%) for two alleles, and only 5 (2.7%) for three alleles. Conclusion Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.

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 The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

2011 ◽  
Vol 78 (4) ◽  
pp. 1063-1068 ◽  
Author(s):  
A. Billard ◽  
V. Laval ◽  
S. Fillinger ◽  
P. Leroux ◽  
H. Lachaise ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Amplification Refractory Mutation System ◽  
Specific Probe ◽  
Monitoring Methods ◽  
Single Nucleotide ◽  
Allele Specific ◽  
Amplification Assay ◽  
Pcr Method

ABSTRACTThe evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

scholarly journals Evaluation of the inhibitory effects of drugs on the growth of Babesia Parasites using real-time PCR method

2020 ◽  
Author(s):  
Xiao-hu Zhai ◽  
Xiao-xiao Feng ◽  
Xian Wu ◽  
Wei-hua He ◽  
Yan-yan Li ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rna Extraction ◽  
Relative Quantification ◽  
Inhibitory Effects ◽  
Diminazene Aceturate ◽  
Pcr Method ◽  
Method Analysis

AbstractIn order to evaluate the inhibitory effects of drug on the growth of babesia parasite, relative quantification real-time PCR method was developed in this study. The 18S rRNA gene was used as target gene for the 2−ΔΔCt method analysis. Meanwhile, Chicken RNA was added into the parasitized blood for total RNA extraction. The β-actin gene of chicken was selected as internal control gene for the 2−ΔΔCt method analysis. Parasitized blood 100 μL, 50 μL, 25μL, 12.5 μL, 6.25 μL was prepared for B. gibsoni relative quantification. Regression analysis results revealed that significant linear relationships between the relative quantification value and parasitemia. The 18S rRNA gene expression was significantly decreased after the treatment of Diminazene aceturate and Artesunate in vitro drug sensitivity test. It suggested that this relative quantification real-time PCR method can be used in evaluating the effects of inhibitory of drug.

Detection of Non-Tuberculosis Mycobacteria by Real Time PCR

2021 ◽  
Vol 19 ◽  
Author(s):  
Mina Ahmadi ◽  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Respiratory Infections ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Beta Actin ◽  
Pcr Method ◽  
Slow Growing

Background: Identification of non-tuberculosis mycobacteria by culture and phenotypic description is commonly used; however, it takes 4 to 6 weeks or even a longer time for slow growing species as well as for identification of some species that may be missed by biochemical characteristics methods. This study aimed to evaluate Real Time PCR for Detection of NTM by Amplification of Internal Transcribed Spacer (ITS) and 16S rRNA. Methods: In our investigation, using Real Time PCR and two pairs of unique primers targeted to ITS and 16S rRNA genes as well as Beta- actin as an internal control, Non tuberculosis mycobacteria species were detected. Results: Real time PCR was performed on the prepared dilutions. In addition, the threshold of sensitivity in this study was 10pg. To test the specificity, the genome of several bacteria responsible for respiratory infections was used, in which only the test response related to the non-tuberculosis mycobacterium genome and internal control was positive. Conclusion: In this research, an effective and up-to-date Real Time PCR method was used to design a diagnostic kit from all aspects. To avoid any error or mistake and to minimize the false results, internal control was used. The ability to design diagnostic kits allows us to increase efficiency, minimize mistakes, and save a considerable amount of time and cost.

A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control

2014 ◽  
Vol 60 (1) ◽  
pp. 11-15 ◽  
Author(s):  
Ersin Karataylı ◽  
Yasemin Çelik Altunoğlu ◽  
Senem Ceren Karataylı ◽  
S. Gökçe K. Alagöz ◽  
Kubilay Çınar ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Virus Rna ◽  
One Step ◽  
Pcr Method ◽  
Armored Rna ◽  
Delta Virus

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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