Molecular characterization and bioinformatics analysis of Ncoa7B, a novel ovulation-associated and reproduction system-specific Ncoa7 isoform

In the present work, we employed bioinformatics search tools to select ovulation-associated cDNA clones with a preference for those representing putative novel genes. Detailed characterization of one of these transcripts, 6C3, by real-time PCR and RACE analyses led to identification of a novel ovulation-associated gene, designatedNcoa7B. This gene was found to exhibit a significant homology to theNcoa7gene that encodes a conserved tissue-specific nuclear receptor coactivator. UnlikeNcoa7,Ncoa7Bpossesses a unique and highly conserved exon at the 5′ end and encodes a protein with a unique N-terminal sequence. Extensive bioinformatics analysis has revealed thatNcoa7Bhas one identifiable domain, TLDc, which has recently been suggested to be involved in protection from oxidative DNA damage. An alignment of TLDc domain containing proteins was performed, and the closest relative identified wasOXR1, which also has a corresponding, highly related short isoform, with just a TLDc domain. Moreover,Ncoa7Bexpression, as seen to date, seems to be restricted to mammals, while other TLDc family members have no such restriction. Multiple tissue analysis revealed that unlikeNcoa7, which was abundant in a variety of tissues with the highest expression in the brain,Ncoa7BmRNA expression is restricted to the reproductive system organs, particularly the uterus and the ovary. The ovarian expression ofNcoa7Bwas stimulated by human chorionic gonadotropin. Additionally, using real-time PCR, we demonstrated the involvement of multiple signaling pathways forNcoa7Bexpression on preovulatory follicles.

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

Annals of Laboratory Medicine ◽

10.3343/alm.2015.35.3.306 ◽

2015 ◽

Vol 35 (3) ◽

pp. 306-313 ◽

Cited By ~ 10

Author(s):

Abdullah Kilic ◽

Mohammad J. Alam ◽

Naradah L. Tisdel ◽

Dhara N. Shah ◽

Mehmet Yapar ◽

...

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Real Time Pcr ◽

Stool Samples ◽

Pcr Method ◽

Simultaneous Identification

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Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0003469 ◽

2015 ◽

Vol 9 (1) ◽

pp. e0003469 ◽

Cited By ~ 16

Author(s):

Robin H. Miller ◽

Clifford O. Obuya ◽

Elizabeth W. Wanja ◽

Bernhards Ogutu ◽

John Waitumbi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Novel Species ◽

Plasmodium Ovale ◽

Pcr Assay ◽

Western Kenya ◽

Plasmodium Ovale Curtisi ◽

Species Specific

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Isolation and molecular characterization of Mycobacterium tuberculosis complex isolated from raw milk in some dairy farms in Egypt

International Journal of Basic and Applied Sciences ◽

10.14419/ijbas.v5i2.5299 ◽

2016 ◽

Vol 5 (2) ◽

pp. 105

Author(s):

Heba Hussien ◽

Eman Mahrous

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Raw Milk ◽

Accurate Method ◽

Tuberculosis Complex ◽

Milk Samples ◽

Source Of Infection

This study was conducted to detect Mycobacterium tuberculosis complex in milk in three Egyptian Governorates; El-Sharkia, El-Menoufia and El-Behera Governorates. 300 milk samples were collected from tuberculin positive cases, 18 (6.0%) were shedding Mycobacterium tuberculosis complex in their milk which detected by real time PCR. On another hand, 170 milk samples were collected from tuberculin negative cases, 5 (2.9%) were shedding Mycobacterium tuberculosis complex in their milk which detected by real time PCR. All milk samples were examined by three techniques including Microscopic examination, culture and real time PCR. Real time PCR is more rapid and accurate method than microscopic and culture method. The isolated colonies from culture were examined by Multiplex PCR to demonstrate the source of infection either human or animal source.

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Detection and Characterization of Circulating Tumor Cells by Quantitative Real-Time PCR

Methods in Molecular Biology - Quantitative Real-Time PCR ◽

10.1007/978-1-4939-9833-3_11 ◽

2019 ◽

pp. 139-151 ◽

Cited By ~ 1

Author(s):

Francesca Salvianti ◽

Filomena Costanza ◽

Gemma Sonnati ◽

Pamela Pinzani

Keyword(s):

Real Time ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Real Time Pcr ◽

Quantitative Real Time Pcr

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Characterization of a universal screening approach for congenital CMV infection based on a highly-sensitive, quantitative, multiplex real-time PCR assay

PLoS ONE ◽

10.1371/journal.pone.0227143 ◽

2020 ◽

Vol 15 (1) ◽

pp. e0227143

Author(s):

Angela Nagel ◽

Emmanouela Dimitrakopoulou ◽

Norbert Teig ◽

Peter Kern ◽

Thomas Lücke ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Universal Screening ◽

Cmv Infection ◽

Pcr Assay ◽

Screening Approach ◽

Highly Sensitive ◽

Congenital Cmv Infection ◽

Congenital Cmv

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A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

Journal of Clinical Microbiology ◽

10.1128/jcm.01986-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e01986-20

Author(s):

Ibne Karim M. Ali ◽

Shantanu Roy

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Small Subunit ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Rdna Sequences ◽

Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

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Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products

Critical Reviews in Food Science and Nutrition ◽

10.1080/10408398.2017.1375893 ◽

2017 ◽

Vol 59 (3) ◽

pp. 423-442 ◽

Cited By ~ 8

Author(s):

Caterina Agrimonti ◽

Benedetta Bottari ◽

Maria Luisa Savo Sardaro ◽

Nelson Marmiroli

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Food Products ◽

Microbial Populations ◽

Dairy Food

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Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.04137-13 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3086-3094 ◽

Cited By ~ 115

Author(s):

Hyatt C. Green ◽

Richard A. Haugland ◽

Manju Varma ◽

Hana T. Millen ◽

Mark A. Borchardt ◽

...

Keyword(s):

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Fecal Pollution ◽

Qpcr Assay ◽

Taqman Assay ◽

Reference Database ◽

Quantitative Real Time Pcr ◽

Ambient Surface

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

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