scholarly journals Microbiological testing of clinical samples before and after periodontal treatment. A comparative methodological study between real‐time PCR and real‐time‐PCR associated to propidium monoazide

2021 ◽  
Author(s):  
Maria Sereti ◽  
Alkisti Zekeridou ◽  
Jose Cancela ◽  
Andrea Mombelli ◽  
Catherine Giannopoulou
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Periodontal Treatment ◽  
Clinical Samples ◽  
Propidium Monoazide ◽  
Methodological Study ◽  
Before And After ◽  
Microbiological Testing

scholarly journals Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR

2004 ◽  
Vol 50 (2) ◽  
pp. 306-312 ◽  
Author(s):  
Stefan S Biel ◽  
Andreas Nitsche ◽  
Andreas Kurth ◽  
Wolfgang Siegert ◽  
Muhsin Özel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow ◽  
Real Time ◽  
Bone Marrow Transplant ◽  
Real Time Pcr ◽  
Marrow Transplant ◽  
Test System ◽  
Urine Samples ◽  
Clinical Samples ◽  
Transplant Patients

Abstract Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 106 genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <103 GE/mL and 100% for urine samples containing 109 GE/mL. Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

scholarly journals A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

2013 ◽  
Vol 44 (2) ◽  
pp. 505-510 ◽  
Author(s):  
Aline Padilha Fraga ◽  
Tatiana de Vargas ◽  
Nilo Ikuta ◽  
André Salvador Kazantzi Fonseca ◽  
Álvaro José Celmer ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Gallisepticum ◽  
Clinical Samples ◽  
Mycoplasma Synoviae ◽  
Commercial Poultry

scholarly journals Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

2020 ◽  
Author(s):  
zhenhua Guo ◽  
Kunpeng Li ◽  
Songlin Qiao ◽  
Xinxin Chen ◽  
Ruiguang Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Limit Of Detection ◽  
African Swine Fever ◽  
Economic Losses ◽  
Clinical Samples ◽  
Diagnosis Method ◽  
Pcr Method ◽  
And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

scholarly journals Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

2020 ◽  
Author(s):  
Vu Thuy Duong ◽  
Le Thi Phuong Tu ◽  
Ha Thanh Tuyen ◽  
Le Thi Quynh Nhi ◽  
James I Campbell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Middle Income ◽  
Middle Income Countries ◽  
Virulence Markers ◽  
Low Middle Income Countries ◽  
High Prevalence ◽  
Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

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

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
Author(s):  
Hanah Kim ◽  
Mina Hur ◽  
Eunsin Bae ◽  
Kyung-A Lee ◽  
Woo-In Lee
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Nucleic Acid Amplification ◽  
Coefficient Of Determination ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Cobas Taqman ◽  
Limit Of Quantitation ◽  
Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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