Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

2003 ◽  
Vol 38 (5) ◽  
pp. 518-524 ◽  
Author(s):  
Juan M Morillo ◽  
Laura Lau ◽  
Mariano Sanz ◽  
David Herrera ◽  
Augusto Silva
Keyword(s):  
Real Time ◽  
Porphyromonas Gingivalis ◽  
Real Time Pcr ◽  
Gene Sequence ◽  
Single Copy ◽  
Actinobacillus Actinomycetemcomitans ◽  
Single Copy Gene ◽  
Quantitative Real Time Pcr ◽  
Copy Gene

scholarly journals Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

2021 ◽  
Vol 9 (8) ◽  
pp. 1610
Author(s):  
Christian Klotz ◽  
Elke Radam ◽  
Sebastian Rausch ◽  
Petra Gosten-Heinrich ◽  
Toni Aebischer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gastrointestinal Disease ◽  
Single Copy ◽  
Marker Genes ◽  
Small Rodent ◽  
Analytical Sensitivity ◽  
Single Copy Gene ◽  
Wild Rodents ◽  
Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

The utility and application of real-time PCR and FISH in the detection of single-copy gene targets inEscherichia coliO157:H7 andSalmonellaTyphimurium

2010 ◽  
Vol 56 (3) ◽  
pp. 254-262 ◽  
Author(s):  
Merriam Haffar ◽  
Kimberley A. Gilbride
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Receptor Gene ◽  
Detection Limits ◽  
Single Copy ◽  
Target Cells ◽  
Single Copy Gene ◽  
Gene Copies ◽  
Copy Gene

The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 × 103invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.

scholarly journals Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens

2004 ◽  
Vol 31 (12) ◽  
pp. 1054-1060 ◽  
Author(s):  
Juan Manuel Morillo ◽  
Laura Lau ◽  
Mariano Sanz ◽  
David Herrera ◽  
Conchita Martin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Gene Sequence ◽  
Single Copy ◽  
Single Copy Gene ◽  
Periodontal Pathogens ◽  
Chain Reaction ◽  
Copy Gene ◽  
Polymerase Chain

scholarly journals Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR

2009 ◽  
Vol 15 (4) ◽  
pp. 195-204 ◽  
Author(s):  
Kati Hyvärinen ◽  
Saara Laitinen ◽  
Susanna Paju ◽  
Anne Hakala ◽  
Liisa Suominen-Taipale ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Single Copy Gene ◽  
Periodontal Pathogens ◽  
Copy Gene ◽  
Detection And Quantification

scholarly journals Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.)

2020 ◽  
Author(s):  
Zheng Yang ◽  
Kai Wang ◽  
Usman Aziz ◽  
Cuizhu Zhao ◽  
Meng Zhang
Keyword(s):  
Real Time ◽  
Avena Sativa ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Expression Patterns ◽  
Single Copy ◽  
Initiation Factor ◽  
Pcr Analysis ◽  
Systematic Research ◽  
Quantitative Real Time Pcr

Abstract Background: Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids.Results: In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for “all samples”, “shoots and roots of seedlings”, “developing seeds” and “developing endosperms” were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets.Conclusions: Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

scholarly journals Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.)

2020 ◽  
Author(s):  
Zheng Yang ◽  
Kai Wang ◽  
Cuizhu Zhao ◽  
Meng Zhang
Keyword(s):  
Real Time ◽  
Avena Sativa ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Single Copy ◽  
Initiation Factor ◽  
Pcr Analysis ◽  
Systematic Research ◽  
Quantitative Real Time Pcr ◽  
Oat Seeds

Abstract Background: Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, none of suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented in oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids.Results: In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for all samples, shoots and roots of seedlings, developing seeds and developing endosperms were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively, among which UBC21 was a four-copy duplicated gene. The reliability was validated by the expression pattern of AsPKP1 (Plastidial Pyruvate Kinase 1) normalized to the most and the least stable RGs in developing oat seeds and endosperms.Conclusions: These results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

Determination of Cry1Ac copy number in transgenic pigeonpeaplants using quantitative real time PCR.

2016 ◽  
Author(s):  
Meenakshi . Jain ◽  
Surender . Khatodia ◽  
Pushpa . Kharb ◽  
Praveen . Batra ◽  
Vijay K. Chowdhury
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Cost Effective ◽  
Single Copy ◽  
Quantitative Real Time Pcr ◽  
Real Time Quantitative Pcr ◽  
Specificity And Sensitivity ◽  
Transgenic Lines ◽  
Pcr Method

Copy number of Cry1Ac in transgenic pigeonpea plants was determined by quantitative real time PCR using Syber Green as fluorescence indicator. Gene specific primers designed to amplify relatively long amplicons (400 – 600 bp), for Cry1Ac was used to increase specificity and sensitivity of Real time PCR. Estimated copy number in transgenic lines using real-time quantitative PCR and southern hybridization was correlated and found to be same i.e. single copy number. This study shows effectivness of real-time PCR method for estimating the transgene copy number in transgenic pigeonpea plants by a simple, accurate and cost effective manner.Keywords: Copy number, Cry1Ac, Pigeonpea transformation, Real-time PCR, Syber green.

scholarly journals Evaluation of duplicated reference genes for quantitative real-time PCR analysis in genome unknown hexaploid oat (Avena sativa L.)

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Zheng Yang ◽  
Kai Wang ◽  
Usman Aziz ◽  
Cuizhu Zhao ◽  
Meng Zhang
Keyword(s):  
Real Time ◽  
Avena Sativa ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Expression Patterns ◽  
Single Copy ◽  
Initiation Factor ◽  
Pcr Analysis ◽  
Systematic Research ◽  
Quantitative Real Time Pcr

Abstract Background Oat (Avena sativa L.), a hexaploid crop with unknown genome, has valuable nutritional, medicinal and pharmaceutical uses. However, no suitable RGs (reference genes) for qPCR (quantitative real-time PCR) has been documented for oat yet. Single-copy gene is often selected as RG, which is challengeable or impactable in unexplored polyploids. Results In this study, eleven candidate RGs, including four duplicated genes, were selected from oat transcriptome. The stability and the optimal combination of these candidate RGs were assessed in 18 oat samples by using four statistical algorithms including the ΔCt method, geNorm, NormFinder and BestKeeper. The most stable RGs for “all samples”, “shoots and roots of seedlings”, “developing seeds” and “developing endosperms” were EIF4A (Eukaryotic initiation factor 4A-3), UBC21 (Ubiquitin-Conjugating Enzyme 21), EP (Expressed protein) and EIF4A respectively. Among these RGs, UBC21 was a four-copy duplicated gene. The reliability was validated by the expression patterns of four various genes normalized to the most and the least stable RGs in different sample sets. Conclusions Results provide a proof of concept that the duplicated RG is feasible for qPCR in polyploids. To our knowledge, this study is the first systematic research on the optimal RGs for accurate qPCR normalization of gene expression in different organs and tissues of oat.

Sign in / Sign up

Export Citation Format

Share Document