Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

2004 ◽  
Vol 102 (1-2) ◽  
pp. 55-65 ◽  
Author(s):  
C DUBOSSON
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Hyopneumoniae ◽  
Clinical Samples ◽  
Pcr Assays

scholarly journals Real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

2003 ◽  
Author(s):  
C. R. Dubosson ◽  
C. Conzelmann ◽  
W. Zimmermann ◽  
H. Häni ◽  
R. Miserez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mycoplasma Hyopneumoniae ◽  
Clinical Samples ◽  
Pcr Assays

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 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.

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 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 Comparative Evaluation of Conventional and Real-Time PCR Assays for Detecting Bacteroides fragilis in Clinical Samples

2013 ◽  
Vol 51 (5) ◽  
pp. 1593-1595 ◽  
Author(s):  
J. Papaparaskevas ◽  
V. Mela ◽  
D. P. Houhoula ◽  
A. Pantazatou ◽  
G. L. Petrikkos ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Comparative Evaluation ◽  
Bacteroides Fragilis ◽  
Clinical Samples ◽  
Pcr Assays

scholarly journals Real-Time PCR Assays for the Specific Detection of Field Balkan Strains of Lumpy Skin Disease Virus

2016 ◽  
Vol 66 (4) ◽  
pp. 444-454 ◽  
Author(s):  
Dejan Vidanović ◽  
Milanko Šekler ◽  
Tamaš Petrović ◽  
Zoran Debeljak ◽  
Nikola Vasković ◽  
...  
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Real Time Pcr ◽  
Skin Disease ◽  
Standard Procedure ◽  
Clinical Samples ◽  
Specific Detection ◽  
Lumpy Skin Disease ◽  
Lumpy Skin Disease Virus ◽  
Pcr Assays

Abstract Lumpy skin disease (LSD) is an important disease of cattle which is included in the OIE list of notifiable terrestrial animal diseases because of its great economic importance. The etiological agent is the Lumpy skin disease virus (LSDV). In the control of LSD attenuated strains of LSDV and SPPV are successfully used as vaccine strains in infected areas. In the case of vaccination policy, due to the possibility of mild or systemic post-vaccination reactions in vaccinated animals, the application of diagnostic procedures that will rapidly and specifically differentiate LSDV field strains from LSD vaccine virus strains are extremely important. Rapidity in diagnostics and disposal of infected animals is one of the key factors in the prevention of spreading the disease. In the presented study we have described the development and validation of two real-time TaqMan-PCR assays for a rapid, sensitive and specific detection of the virulent field LSDV strain currently circulating in the Balkan Peninsula. Specificity for the field strain and exclusivity for vaccine strains was tested on 171 samples from naturally infected and vaccinated animals. The results of this study show that both developed real-time PCR assays are more sensitive than the conventional nested PCR in detecting field LSDV strains thus enabling rapid and high-throughput detection of animals infected with field strains of LSDV. In conclusion, both KV-2 and FLI real-time PCR assays described in this study are simple, rapid, sensitive and suitable for routine use in a diagnostic laboratory and have the potential to replace conventional nested gel-based PCR assays as the standard procedure for the detection of field strains of LSDV in clinical samples.

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