scholarly journals Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

2014 ◽  
Vol 11 (1) ◽  
Author(s):  
Alcione de Oliveira dos Santos ◽  
Luan Felipo Botelho Souza ◽  
Lourdes Maria Borzacov ◽  
Juan Miguel Villalobos-Salcedo ◽  
Deusilene Souza Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Amazon Region ◽  
Wide Range ◽  
Pcr Test

scholarly journals Development of a sensitivity and cost-effective real time pcr: measuring a wide range of HBV DNA concentrations

2013 ◽  
Author(s):  
Alcione Santos ◽  
Luan Souza ◽  
Lourdes Borzacov ◽  
Juan Villalobos-Salcedo ◽  
Deusilene Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Wide Range

QUANTITATION OF HBV DNA BY REALTIME PCR FOR HBV DETECTION AND FOLLOW-UP VIRAL LOAD IN PATIENTS WITH CHRONICHBV HEPATITIS USING ANTIVIRAL DRUGS

2011 ◽  
pp. 64-70
Author(s):  
Van An Le ◽  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Antiviral Drugs ◽  
Hbv Dna ◽  
Therapeutic Effects ◽  
Pcr Assay ◽  
Serum Samples ◽  
Wide Range ◽  
Chronic Hbv

Background: Real-time PCR assay has been routinely used in many laboratories for HBV determination and follow –up of the HBV DNA levels in serum of chronic HBV patients during antiviral therapy. We used a commercialized real-time PCR procedure based on TaqMan chemistry to quantify HBV DNA levels from patients with chronic HBV hepatitis for HBV detection and monitoring HBV DNA during antiviral drug using. Materials and method: This study was carried out in 56 patients with chronic HBV hepatitis using antiviral drugs. A commercialized real-time PCR assay based on TaqMan chemistry was used to quantify HBV DNA concentration in double serum samples from each patient, the first sample was collected at the first quantificative testing and the second sample was collected for a follow-up in 6 or 9 months of interval. The assay has a dymnamic range from 3 x 102 copies/ml at minimun level to 1010copies /ml. Sample testing was always run with triple dilutions of standard, the HBV DNA quantitations were analysed by Stratagene software and calculated in number of copies per ml of serum sample. Results: The HBV DNA levels in all the first serum samples had a wide range from 3x102 to 1010 copies/ml, of these first samples there were 30% (17/56) with the highest levels from 108 to 1010copies/ml and there was no sample negative for HBV DNA. With the second serum samples, there were 23,2% (13/56) underdetectable for HBV DNA and the sample percentage with the highest HBV DNA levels was only 5,3% (3/56). The HBV DNA levels at the second serum samples were lower in 44 patients (78%) and were higher in 12 patients (21,4%) in comparision with that of the first samples. The average amounts of HBV DNA decrease in patients using antiviral drugs were 2,3 x 108 copies/ml with adefovir and 4,2 x108 copies/ml with lamivudine, and the average numbers of HBV DNA increase were 1,4 x 108 copies/ml with lamivudine and 7,5 x 107 copies/ml with entecavir. Conclusions: Real-time PCR assay was found to be very useful in quantification of HBV DNA level in chronic HBV patients and also for monitoring the therapeutic effects of antiviral drugs.

scholarly journals Introducing Novel Molecular-based Method for Quantification of Homologous Recombination Efficiency

2021 ◽  
Author(s):  
Mustapha Dibbasey ◽  
Terry Gaymes
Keyword(s):  
Homologous Recombination ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Lacz Gene ◽  
Expression Arrays ◽  
Recombinant Product ◽  
Wide Range ◽  
High Level

AbstractBackgroundHomologous recombination (HR) pathway is a DNA double-stranded breaks repair pathway well-known for its high level of accuracy. Low HR pathway efficiency clinically known as homologous recombination deficiency (HRD) was identified in some cancers such as breast and ovarian cancers and studies have reported the sensitivity of HRD cancer cells to DNA repair inhibitors such as Olaparib. However, current techniques including immunofluorescence-based technique are qualitative-based, hence lack sensitivity to determine the functionality of HR pathway. Additionally, some of the techniques including gene expression arrays require expression study of wide range genes involve in HR pathway, which is not cost-effective. The aim of the study is to optimise a PCR-based assay (Norgen’s Homologous Recombination kit) that can be employed to quantitate HR efficiency in cells, which accurately reflects the functional status of HR pathway.Methods and FindingsThe kit has two test plasmids (dl-1 and dl-2) with partial deletions in the LacZ gene and the plasmids are generated from modification of pUC19. HR-proficient (HeLa and AsPC-1) and HR-deficient (CAPAN-1 cells) cancer cell lines were transfected with the two plasmids to generate functional LacZ gene (i.e. recombinant product). The recombinant product was quantified by real-time PCR. Although recombinant product was generated in all the cell lines, our real-time PCR demonstrated a high quantity of recombinant product in HeLa cell line whilst low quantity in CAPAN-1 and AsPC-1 cell lines. The quantity of recombinant product generated and quantified reflects HR pathway efficiency.ConclusionOverall, the results have provided some evidence that the PCR-based kit can be suitably employed for quantification of HR efficiency provided appropriate transfection method and reagent are used. However, further study is required to confirm HR efficiency status of AsPC-1 cells to ascertain the low HR efficiency detected by the kit in these cells.

scholarly journals In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manash Jyoti Kalita ◽  
Kalpajit Dutta ◽  
Gautam Hazarika ◽  
Ridip Dutta ◽  
Simanta Kalita ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Sybr Green ◽  
Green Approach ◽  
Novel Approach ◽  
Effective Manner ◽  
Wide Range ◽  
Thermal Cycler

AbstractAs the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

scholarly journals Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

2014 ◽  
Vol 14 (1) ◽  
Author(s):  
Runa M Grimholt ◽  
Petter Urdal ◽  
Olav Klingenberg ◽  
Armin P Piehler
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Globin Gene ◽  
Copy Number Variations ◽  
Globin Genes ◽  
Quantitative Real Time Pcr ◽  
Human Genetic Disease ◽  
Large Deletions ◽  
Wide Range

Abstract Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

scholarly journals Cost-effective optimization of real-time PCR-based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

2015 ◽  
Vol 119 (5) ◽  
pp. 1391-1402 ◽  
Author(s):  
M.S.R. Fachmann ◽  
M.H. Josefsen ◽  
J. Hoorfar ◽  
M.T. Nielsen ◽  
C. Löfström
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Polymerases ◽  
Cost Effective

scholarly journals Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Andrew Jenkins ◽  
Cecilie Raasok ◽  
Benedikte N. Pedersen ◽  
Kristine Jensen ◽  
Åshild Andreassen ◽  
...  
Keyword(s):  
Real Time ◽  
Ixodes Ricinus ◽  
Real Time Pcr ◽  
Accession Number ◽  
Northern Limit ◽  
Groel Gene ◽  
Candidatus Neoehrlichia Mikurensis ◽  
Control Plasmid ◽  
Neoehrlichia Mikurensis ◽  
Pcr Test

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

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