scholarly journals Development of Real-time PCR Kit for Detection and Quantitation of Six Common Mutations A3243G, G3380A, A8344G, T8993G, T8993C, G11778A of Human Mitochondrial Genome

2022 ◽  
Author(s):  
Nguyen Thi Hong Loan ◽  
Phung Bao Khanh ◽  
Le Ngoc Anh ◽  
Cao Vu Hung ◽  
Pham Van Anh ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Real Time ◽  
Real Time Pcr ◽  
Mitochondrial Diseases ◽  
Thermal Conditions ◽  
Locked Nucleic Acid ◽  
Specific Primers ◽  
A3243g Mutation ◽  
Selection Of ◽  
Human Mitochondrial Genome

A procedure for production of a real-time PCR kit for detection and quantitation of 6 common mitochondrial genome mutations including A3243G, G3380A, A8344G, T8993G, T8993C, G11778A using fluorescent locked nucleic acid (LNA) Taqman probes was reported. The procedure consists of designing of specific primers and LNA probes, selection of master mixture components and real-time PCR thermal conditions. The produced kit had specificity of 100% and sensitivity ≥ 1% and remained fully active after 7 days of storage at 25 oC or 20 days at 4 oC or 6 months at -20 oC. The kit was used to analyze A3243G, G3380A, A8344G, T8993G, T8993C, G11778A mutations from 69 patients tentatively diagnosed with mitochondrial diseases and 3 cases of A3243G carriers (4.34%) was found. In these cases, the A3243G mutation was heteroplasmic, maternally inherited, and the heteroplasmy level was shown to be related to the symptome expression.tome expression.

scholarly journals Detection of a new case harboring mitochondrial A3243G mutation of MELAS syndrome

2017 ◽  
Vol 33 (1S) ◽  
Author(s):  
Phung Bao Khanh ◽  
Nguyen Minh Hoang ◽  
Pham Van Anh ◽  
Le Ngoc Anh ◽  
Cao Vu Hung ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Lactic Acidosis ◽  
Real Time ◽  
Mutation Analysis ◽  
Real Time Pcr ◽  
Melas Syndrome ◽  
Mitochondrial Encephalopathy ◽  
Pcr Rflp ◽  
A3243g Mutation ◽  
Severity Of The Disease

Mitochondrial genome A3243G mutation in the tRNALeu(UUR)  encodinggene (MTTL)is the main cause of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). This mutation exists in heteroplasmic form and severity of the disease is affected by many factors including heteroplasmy level. In this study, a pediatric proband (female, 8 years old) was found to carry A3243G mutation at 77.6% of heteroplasmy by using PCR-RFLP in combination with real-time PCR. The results of  the A3243G mutation analysis of the proband’s family showed that her mother without any symptoms of encephalopathyalso carried the mutation at 7.9% of heteroplasmy whereas the mutation was not found in the proband’s healthy father and healthy sister, indicating that the proband received the A3243G mutation from her mother and the expression of MELAS syndromes depended on the level of heteroplasmy.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

scholarly journals Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143444 ◽  
Author(s):  
Yong Zhao ◽  
Guilian Li ◽  
Chongyun Sun ◽  
Chao Li ◽  
Xiaochen Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Locked Nucleic Acid Probe

Real-time PCR-assisted selection of wheat plants transformed with HMW glutenin subunit genes

2005 ◽  
Vol 41 (1) ◽  
pp. 133-136 ◽  
Author(s):  
Valeria Terzi ◽  
Gabriela Pastori ◽  
Peter R. Shewry ◽  
Natale Di Fonzo ◽  
A. Michele Stanca ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Glutenin Subunit ◽  
Hmw Glutenin Subunit ◽  
Hmw Glutenin ◽  
Selection Of

scholarly journals Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

BioTechniques ◽  
2005 ◽  
Vol 38 (4) ◽  
pp. 605-610 ◽  
Author(s):  
Lone Hummelshoj ◽  
Lars P. Ryder ◽  
Hans O. Madsen ◽  
Lars K. Poulsen
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Locked Nucleic Acid

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis

2009 ◽  
Vol 129 (1-2) ◽  
pp. 115-118 ◽  
Author(s):  
Yvette M. Schlotter ◽  
Eveline Z. Veenhof ◽  
Bas Brinkhof ◽  
Victor P.M.G. Rutten ◽  
Bart Spee ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Skin Biopsies ◽  
Healthy Dogs ◽  
Selection Of

scholarly journals Detection of Spinal Muscular Atrophy Using a Duplexed Real-Time PCR Approach With Locked Nucleic Acid-Modified Primers

2021 ◽  
Vol 41 (1) ◽  
pp. 101-107
Author(s):  
Jianyan Pan ◽  
Chunhua Zhang ◽  
Yanling Teng ◽  
Sijing Zeng ◽  
Siyi Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Spinal Muscular Atrophy ◽  
Real Time ◽  
Real Time Pcr ◽  
Muscular Atrophy ◽  
Locked Nucleic Acid

scholarly journals Comparison of different conditions for DNA extraction in sputum - a pilot study

2019 ◽  
Vol 14 ◽  
Author(s):  
Martina Oriano ◽  
Leonardo Terranova ◽  
Antonio Teri ◽  
Samantha Sottotetti ◽  
Luca Ruggiero ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Alpha Diversity ◽  
Diversity Indices ◽  
Extraction Yield ◽  
Chronic Respiratory Diseases ◽  
Dna Yield ◽  
Selection Of

Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

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