scholarly journals Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

2008 ◽  
Vol 57 (12) ◽  
pp. 1547-1552 ◽  
Author(s):  
Zhijun Bai ◽  
Licheng Liu ◽  
Zeng Tu ◽  
Lisi Yao ◽  
Jianwei Liu ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Serum Samples ◽  
Serological Tests ◽  
Wide Range ◽  
Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

scholarly journals Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test

2019 ◽  
Vol 39 (4) ◽  
pp. 255-262
Author(s):  
Paula L. Martin ◽  
Nestor O. Stanchi ◽  
Bibiana F. Brihuega ◽  
Estela Bonzo ◽  
Lucía Galli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Urine Samples ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Presumptive Diagnosis ◽  
Microagglutination Test ◽  
Pcr Assays

ABSTRACT: Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

scholarly journals Comparison of Two Commercial Real-Time PCR Assays for Detection of Dengue Virus in Patient Serum Samples

2014 ◽  
Vol 52 (10) ◽  
pp. 3781-3783 ◽  
Author(s):  
J. Saengsawang ◽  
O. Nathalang ◽  
M. Kamonsil ◽  
V. Watanaveeradej
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Serum Samples ◽  
Pcr Assays

scholarly journals Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41

2005 ◽  
Vol 71 (6) ◽  
pp. 3131-3136 ◽  
Author(s):  
Narayanan Jothikumar ◽  
Theresa L. Cromeans ◽  
Vincent R. Hill ◽  
Xiaoyan Lu ◽  
Mark D. Sobsey ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Melting Curve Analysis ◽  
Pcr Assay ◽  
Adenovirus Serotype ◽  
Human Adenoviruses ◽  
Wide Range ◽  
Pcr Assays

ABSTRACT A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.

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 Performance Evaluation of Commercial Dengue Diagnostic Tests for Early Detection of Dengue in Clinical Samples

2017 ◽  
Vol 2017 ◽  
pp. 1-4 ◽  
Author(s):  
Tuan Nur Akmalina Mat Jusoh ◽  
Rafidah Hanim Shueb
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Diagnostic Test ◽  
Rapid Diagnostic Test ◽  
Dengue Infection ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Serum Samples ◽  
Kappa Agreement

The shattering rise in dengue virus infections globally has created a need for an accurate and validated rapid diagnostic test for this virus. Rapid diagnostic test (RDT) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostic detection are useful tools for diagnosis of early dengue infection. We prospectively evaluated the diagnostic performance of nonstructural 1 (NS1) RDT and real-time RT-PCR diagnostic kits in 86 patient serum samples. Thirty-six samples were positive for dengue NS1 antigen while the remaining 50 were negative when tested with enzyme-linked immunosorbent assay (ELISA). Commercially available RDTs for NS1 detection, RTK ProDetect™, and SD Bioline showed high sensitivity of 94% and 89%, respectively, compared with ELISA. GenoAmp® Trioplex Real-Time RT-PCR and RealStar® Dengue RT-PCR tests presented a comparable kappa agreement with 0.722. The result obtained from GenoAmp® Real-Time RT-PCR Dengue test showed that 14 samples harbored dengue virus type 1 (DENV-1), 8 samples harbored DENV-2, 2 samples harbored DENV-3, and 1 sample harbored DENV-4. 1 sample had a double infection with DENV-1 and DENV-2. The NS1 RDTs and real-time RT-PCR tests were found to be a useful diagnostic for early and rapid diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

scholarly journals Evaluation of designed IS711 primers and universal primers of B4 and B5 for detection of Brucella spp. in clinical samples

2019 ◽  
Author(s):  
Pedram Heidari ◽  
Mitra Salehi ◽  
Abbas Akhavan Sepahi ◽  
Mohamad Reza Razavi
Keyword(s):  
Blood Culture ◽  
Universal Primers ◽  
Clinical Samples ◽  
Target Sequence ◽  
Serum Samples ◽  
Serological Tests ◽  
Molecular Approaches ◽  
Bacterial Agent ◽  
Detection And Identification ◽  
Wide Range

Abstract Background Brucellosis as a global concern is a zoonotic infectious disease which affects a wide range of individual in developing countries. A confirmed diagnosis is required to isolate the bacterial agent from clinical specimens like blood, bone marrow, CSF or tissues. Microbiological, serological and molecular approaches are useful for detection and identification of Brucella spp. and blood culture is known as the gold standard for Brucella spp. Diagnosis of brucellosis through polymerase chain reaction (PCR) could be more sensitive and specific than other classical methods such as blood culture and conventional serological tests. Until now different Brucella specific sequences like BCSP 31, IS711 and 16SrRNAwere amplified for detection of Brucella Spp. Results Amplification of extracted DNA from serum of 49 suspected patients were tested with two sets of specific primers. The BCSP31 sequence amplicon was 223 bp and all the 49 (100%) serum specimens isolated from suspected patients were positive by B4 and B5 primers, even the 4 cases out of 49 2ME negative samples were positive. Detection of Brucella in serum samples by designed IS711 primers revealed the amplicon of IS711 with 448 bp length. Among the 49 serum samples isolated from patients, 46 (93.87%) cases were positive. The B4-B5 primers and IS711 designed primer recognized 100% (49/49) and 94% (46/49) of the cases, respectively. Conclusion This study shows that the specificity of the 2 primer sets is 100% and the sensitivity of B4-B5 primers is 100%, while the sensitivity of the designed primers of IS711 is 94%. The B4-B5 primers can detect the least number of both B. melitensis and B. abortus, 0.05 CFU/reaction. However, the designed IS711 set is able to detect 2 CFU/reaction, about 2.5×102 times more sensitive than results of other experiments for detection of IS711 target sequence in the specimens.

SYBR green and TaqMan real-time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections

2006 ◽  
Vol 78 (6) ◽  
pp. 760-763 ◽  
Author(s):  
Alessandra Cristina Gomes-Ruiz ◽  
Renata Teodoro Nascimento ◽  
Sérgio Oliveira de Paula ◽  
Benedito Antônio Lopes da Fonseca
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Virus Type ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Dengue Virus Type 3 ◽  
Pcr Assays ◽  
Type 3

scholarly journals Comparison of ePlex Respiratory Pathogen Panel with Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Pathogens

2017 ◽  
Vol 55 (6) ◽  
pp. 1938-1945 ◽  
Author(s):  
R. H. T. Nijhuis ◽  
D. Guerendiain ◽  
E. C. J. Claas ◽  
K. E. Templeton
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Time Frame ◽  
Respiratory Pathogen ◽  
Clinical Samples ◽  
Respiratory Pathogens ◽  
Clinical Specimens ◽  
Hands On ◽  
Pcr Assays ◽  
Short Time

ABSTRACT Infections of the respiratory tract can be caused by a diversity of pathogens, both viral and bacterial. Rapid microbiological diagnosis ensures appropriate antimicrobial therapy as well as effective implementation of isolation precautions. The ePlex respiratory pathogen panel (RP panel) is a novel molecular biology-based assay, developed by GenMark Diagnostics, Inc. (Carlsbad, CA), to be performed within a single cartridge for the diagnosis of 25 respiratory pathogens (viral and bacterial). The objective of this study was to compare the performance of the RP panel with those of laboratory-developed real-time PCR assays, using a variety of previously collected clinical respiratory specimens. A total of 343 clinical specimens as well as 29 external quality assessment (EQA) specimens and 2 different Middle East respiratory syndrome coronavirus isolates have been assessed in this study. The RP panel showed an agreement of 97.4% with the real-time PCR assay regarding 464 pathogens found in the clinical specimens. All pathogens present in clinical samples and EQA samples with a threshold cycle ( C T ) value of <30 were detected correctly using the RP panel. The RP panel detected 17 additional pathogens, 7 of which could be confirmed by discrepant testing. In conclusion, this study shows excellent performance of the RP panel in comparison to real-time PCR assays for the detection of respiratory pathogens. The ePlex system provided a large amount of useful diagnostic data within a short time frame, with minimal hands-on time, and can therefore potentially be used for rapid diagnostic sample-to-answer testing, in either a laboratory or a decentralized setting.

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