Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

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Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae)

PLoS ONE ◽

10.1371/journal.pone.0181862 ◽

2017 ◽

Vol 12 (7) ◽

pp. e0181862 ◽

Cited By ~ 15

Author(s):

Ya-Wen Chang ◽

Jing-Yun Chen ◽

Ming-Xing Lu ◽

Yuan Gao ◽

Zi-Hua Tian ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Pcr Analysis ◽

Liriomyza Trifolii ◽

Quantitative Real Time Pcr ◽

Experimental Conditions

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Real-time PCR analysis of the apoptosis related genes in ATRA treated APL t(15;17) patients

Experimental & Molecular Medicine ◽

10.1038/emm.2003.59 ◽

2003 ◽

Vol 35 (5) ◽

pp. 454-459 ◽

Cited By ~ 5

Author(s):

Hakan Savli ◽

Sema Sirma ◽

Balint Nagy ◽

Melih Aktan ◽

Guncag Dincol ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Analysis

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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

10.21203/rs.3.rs-59710/v2 ◽

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.

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Touchable 3D hierarchically structured polyaniline nanoweb for capture and detection of pathogenic bacteria

Nano Convergence ◽

10.1186/s40580-021-00280-9 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Kyung Hoon Kim ◽

MinHo Yang ◽

Younseong Song ◽

Chi Hyun Kim ◽

Young Mee Jung ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pathogenic Bacteria ◽

Bacterial Pathogens ◽

Physical Contact ◽

Structural Deformation ◽

Pcr Analysis ◽

Mechanical Resistance ◽

Fluorescence Signal ◽

The Real

AbstractA bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

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