scholarly journals More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ka-Cheung Luk ◽  
Jeffrey Gersch ◽  
Barbara J. Harris ◽  
Vera Holzmayer ◽  
Dora Mbanya ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Splice Variant ◽  
Splice Variants ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr ◽  
Viral Loads ◽  
Hbv Genotypes ◽  
Dna And Rna

AbstractHBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

scholarly journals DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

2016 ◽  
Vol 3 (1) ◽  
pp. 7 ◽  
Author(s):  
Michael Lebuhn ◽  
Jaqueline Derenkó ◽  
Antje Rademacher ◽  
Susanne Helbig ◽  
Bernhard Munk ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Interlaboratory Comparison ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Dna And Rna

scholarly journals Real-Time PCR Detection Patterns of Porcine Circovirus Type 2 (PCV2) in Polish Farms with Different Statuses of Vaccination against PCV2

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1135 ◽  
Author(s):  
Aleksandra Woźniak ◽  
Dagmara Miłek ◽  
Piotr Matyba ◽  
Tomasz Stadejek
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Quantitative Real Time Pcr ◽  
Viral Loads ◽  
Oral Fluids ◽  
Significant Difference

Porcine circovirus type 2 (PCV2) is a globally spread pathogen controlled with generally highly efficacious vaccination protocols. In order to compare PCV2 detection profiles in farms with different vaccination statuses, serum (359) and fecal pools (351) and oral fluids (209) from four farms that do not vaccinate against PCV2 (NON-VAC) and from 22 farms that do vaccinate (VAC) were tested with quantitative real-time PCR. Additionally, nucleotide sequences of ORF2 of the virus were obtained from selected samples. Three genotypes, PCV2a, PCV2b, and PCV2d, were detected. Significant differences (p < 0.05) in PCV2 prevalence and quantities between the VAC and NON-VAC farms were evident. In five VAC farms, no viremia or shedding in feces was detected. On the other hand, in four VAC farms, the results were very similar to those from NON-VAC farms. No significant difference in PCV2 prevalence in oral fluids was observed between VAC and NON-VAC farms. An examination of viremia can be recommended for the detection of vaccination efficacy issues. The median of the PCV2 viral loads >6.0 log10 copies/mL in pooled sera from the vaccinated population should be considered a very strong indication that the vaccination protocol needs revision.

Detection of Cryptosporidium parvum and Giardia lamblia in Water Supply by Quantitative Real-Time PCR

2012 ◽  
Vol 518-523 ◽  
pp. 3707-3711
Author(s):  
Peng Peng ◽  
Rui Bao Jia ◽  
Yu Mei Liu ◽  
Li Li
Keyword(s):  
Water Supply ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Pcr Primers ◽  
Quantitative Real Time Pcr ◽  
Rt Pcr ◽  
High Recovery ◽  
Pcr Method

Cryptosporidium parvum and Giardia lamblia are common pathogenic protozoa in water, which pose high risk to drinking water supply. In the present study, detection of C. parvum and G. lamblia was performed by quantitative real-time PCR (RT-PCR). Pairs of PCR primers were evaluated for the detection specificity to pathogenic C. parvum and G. lamblia. The recovery of the RT-PCR detection procedure was examined and high recovery rates (i.e., more than 45％ for C. parvum and more than 50％ for G. lamblia ) were achieved. The RT-PCR method was used to detect C. parvum and G. lamblia in a secondary water supply. The results indicated the potential application of the quantitative RT- PCR method in detection of C. parvum and G. lamblia in water supply.

scholarly journals Determination of Viral Load by Quantitative Real-Time PCR in Herpes Simplex Encephalitis Patients

Intervirology ◽  
2014 ◽  
Vol 57 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Shradha S. Bhullar ◽  
Nitin H. Chandak ◽  
Hemant J. Purohit ◽  
Girdhar M. Taori ◽  
Hatim F. Daginawala ◽  
...  
Keyword(s):  
Viral Load ◽  
Herpes Simplex ◽  
Real Time ◽  
Real Time Pcr ◽  
Herpes Simplex Encephalitis ◽  
Quantitative Real Time Pcr

scholarly journals Whatman FTA cards versus plasma specimens for the quantitation of HIV-1 RNA using two real-time PCR assays

2020 ◽  
Vol 2 (8) ◽  
Author(s):  
Abdourahamane Yacouba ◽  
Malika Congo ◽  
Gérard Komonsira Dioma ◽  
Hermann Somlare ◽  
David Coulidiaty ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Filter Paper ◽  
Real Time Pcr ◽  
Load Testing ◽  
Viral Loads ◽  
Viral Load Testing ◽  
Fta Cards ◽  
Pcr Assays ◽  
Hiv 1

Background. Several studies have compared the use of dried blot spot (DBS) as an alternative to plasma specimens, mainly using Whatman 903 cards as filter paper. The aim of this study was to evaluate the use of Whatman FTA card (FTA card) specimens for HIV-1 viral load testing compared to plasma specimens using two real-time PCR assays manufactured by Roche and Abbott. Methodology. A cross-sectional study was conducted between April 2017 and September 2017 on HIV-1 patients admitted to Yalgado Ouédraogo Teaching Hospital. Paired FTA cards and plasma specimens were collected and analysed using the Abbott Real-Time HIV-1 assay (Abbott) and COBAS AmpliPrep/COBAS TaqMan v2.0 (Roche). Results. In total, 107 patients were included. No statistical differences (P>0.05) were observed between the mean viral loads obtained from the FTA cards and those of the plasma specimens using the Roche and Abbott assays. In total, 29 samples with Roche and 15 samples with Abbott assay showed discrepant results. At viral loads of ≤1000 copies ml−1, the sensitivity and specificity of the FTA cards were 78.6 and 100% with Roche, and 92.3 and 95.9% with Abbott, respectively. Both the Roche and Abbott assays showed good correlation and agreement between the FTA cards and plasma values. Conclusion. Our study demonstrates the feasibility of using FTA card filter paper for HIV-1 viral load testing. However, further studies will be required for the validation of the use of FTA card filter paper in HIV-1 treatment monitoring.

Detecting and monitoring endophytic colonization by Pseudomonas putida BP25 in black pepper (Piper nigrum L.) using quantitative real-time PCR

2017 ◽  
Vol 26 (1) ◽  
pp. 01 ◽  
Author(s):  
V N Agisha ◽  
S J Eapen ◽  
R S Bhai ◽  
A Kumar
Keyword(s):  
Pseudomonas Putida ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Colonization ◽  
Phytophthora Capsici ◽  
Pcr Primers ◽  
Black Pepper ◽  
Piper Nigrum ◽  
Quantitative Real Time Pcr ◽  
Athelia Rolfsii

A quantitative real-time PCR assay was developed to quantify Pseudomonas putida BP25, an antagonistic endophyte against a broad range of pathogens in black pepper such as Phytophthora capsici, Colletotrichum gloeosporioides, Rhizoctonia solani, Gibberella moniliformis, Athelia rolfsii and a plant parasitic nematode, Radopholus similis. The real-time PCR primers were designed based on the16S rRNA sequences of P. putida strains and specificity of the primers was confirmed. The detection limit of the assay was found to be 1 pg. The assay detected and quantified the bacterial colonization in the roots at weekly intervals after inoculation. The P. putida DNA was quantified to be 0.4 ng in roots corresponding to 5.4 log10 CFU g-1 at 7th and 14th day after inoculation (DAI). A decline in endophyte population was observed during 21st and 28th DAI and the DNA concentration ranged from 3.7-4.6 pg corresponding to 3.4-3.5 log10 CFU g-1 of root. No amplification could be obtained in stem and leaf samples. The newly developed real-time PCR could be useful for detection, quantification and monitoring of endophytic P. putida BP25 in different plant tissues.  

scholarly journals Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

2007 ◽  
Vol 73 (7) ◽  
pp. 2173-2179 ◽  
Author(s):  
Kerttu Koskenniemi ◽  
Christina Lyra ◽  
Pirjo Rajaniemi-Wacklin ◽  
Jouni Jokela ◽  
Kaarina Sivonen
Keyword(s):  
Baltic Sea ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
The Baltic Sea ◽  
Gene Copies ◽  
Qpcr Method ◽  
The Baltic

ABSTRACT A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml−1 of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.

scholarly journals Anchor-Based Fluorescent Amplicon Generation Assays (FLAG) for Real-Time Measurement of Human Cytomegalovirus, Epstein–Barr Virus, and Varicella-Zoster Virus Viral Loads

2008 ◽  
Vol 54 (11) ◽  
pp. 1900-1907 ◽  
Author(s):  
Alessandro Di Nicola ◽  
Elisa Ghezzi ◽  
Federico Gillio ◽  
Francesco Zerilli ◽  
Erlet Shehi ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Real Time Pcr ◽  
Epstein Barr Virus ◽  
Varicella Zoster ◽  
Viral Loads ◽  
Barr Virus ◽  
Epstein Barr ◽  
Diagnostic Sensitivity And Specificity

Abstract Background: Monitoring the human cytomegalovirus (HCMV), Epstein–Barr virus (EBV), or varicella-zoster virus (VZV) viral load is an important factor in the management of immunosuppressed patients, such as recipients of solid-organ or bone marrow transplants. The advent of real-time PCR technologies has prompted the widespread development of quantitative PCR assays for the detection of viral loads and other diagnostic purposes. Methods: The fluorescent amplicon generation (FLAG) technology uses the PspGI restriction enzyme to monitor PCR product generation. We modified the FLAG technology by introducing an accessory oligonucleotide “anchor” that stabilizes the binding of the forward primer to the target sequence (a-FLAG). We developed assays for HCMV, EBV, and VZV that incorporated an internal amplification-control reaction to validate negative results and extensively analyzed the performance of the HCMV a-FLAG assay. Results: The 3 assays performed similarly with respect to reaction efficiency and linear range. Compared with a commercially available kit, the HCMV a-FLAG assay results showed good correlation with calculated concentrations (r = 0.9617), excellent diagnostic sensitivity and specificity (99% and 95%, respectively), and similar values for the linear range (1–107 copies/μL), analytical sensitivity (0.420 copies/μL), and intra- and interassay imprecision. Conclusions: The a-FLAG assay is an alternative real-time PCR technology suitable for detecting and quantifying target-DNA sequences. For clinical applications such as the measurement of viral load, a-FLAG assays provide multiplex capability, internal amplification control, and high diagnostic sensitivity and specificity.

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