scholarly journals Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine

2006 ◽  
Vol 72 (11) ◽  
pp. 7148-7155 ◽  
Author(s):  
Núria Hierro ◽  
Braulio Esteve-Zarzoso ◽  
Ángel González ◽  
Albert Mas ◽  
Jose M. Guillamón
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
Yeast Extract Peptone Dextrose ◽  
Different Strains ◽  
26S Rrna ◽  
First Time

ABSTRACT Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

scholarly journals Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay

2008 ◽  
Vol 46 (8) ◽  
pp. 2547-2554 ◽  
Author(s):  
I. Gutierrez-Aguirre ◽  
A. Steyer ◽  
J. Boben ◽  
K. Gruden ◽  
M. Poljsak-Prijatelj ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Sensitive Detection ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Quantitative Pcr Assay

scholarly journals Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

2004 ◽  
Vol 70 (4) ◽  
pp. 2373-2382 ◽  
Author(s):  
Torben L. Skovhus ◽  
Niels B. Ramsing ◽  
Carola Holmström ◽  
Staffan Kjelleberg ◽  
Ingela Dahllöf
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Green Alga ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Marine Animals ◽  
Real Time Quantitative Pcr ◽  
Marine Samples ◽  
Marine Habitats ◽  
Pcr Method

ABSTRACT A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

scholarly journals Variability of the Reverse Transcription Step: Practical Implications

2015 ◽  
Vol 61 (1) ◽  
pp. 202-212 ◽  
Author(s):  
Stephen Bustin ◽  
Harvinder S Dhillon ◽  
Sara Kirvell ◽  
Christina Greenwood ◽  
Michael Parker ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Molecular Diagnostic ◽  
Published Data ◽  
Real Time Quantitative Pcr ◽  
Mrna Targets ◽  
High Concentrations ◽  
Diagnostic Applications ◽  
Practical Implications

Abstract BACKGROUND The reverse transcription (RT) of RNA to cDNA is a necessary first step for numerous research and molecular diagnostic applications. Although RT efficiency is known to be variable, little attention has been paid to the practical implications of that variability. METHODS We investigated the reproducibility of the RT step with commercial reverse transcriptases and RNA samples of variable quality and concentration. We quantified several mRNA targets with either singleplex SYBR Green I or dualplex probe-based reverse transcription real-time quantitative PCR (RT-qPCR), with the latter used to calculate the correlation between quantification cycles (Cqs) of mRNA targets amplified in the same real-time quantitative PCR (qPCR) assay. RESULTS RT efficiency is enzyme, sample, RNA concentration, and assay dependent and can lead to variable correlation between mRNAs from the same sample. This translates into relative mRNA expression levels that generally vary between 2- and 3-fold, although higher levels are also observed. CONCLUSIONS Our study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA. Variability can be minimized by choosing an appropriate RTase and high concentrations of RNA and characterizing the variability of individual assays by use of multiple RT replicates.

scholarly journals Effect of Ag Nanoparticles on Denitrification and Microbial Community in a Paddy Soil

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao Zhang ◽  
Di Dang ◽  
Lingsi Zheng ◽  
Lingyu Wu ◽  
Yu Wu ◽  
...  
Keyword(s):  
Microbial Community ◽  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Paddy Soil ◽  
Ag Nanoparticles ◽  
High Throughput Sequencing ◽  
Emission Rate ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr

The extensive application of Ag nanoparticles (AgNPs) in industry, agriculture, and food processing areas increases the possibility of its release and accumulation to agroecosystem, but the effects of AgNPs to denitrification and the microbial community in paddy ecosystems are still poorly studied. In this study, microcosmic simulation experiments were established to investigate the response of soil denitrification to different levels of AgNPs (i.e., 0.1, 1, 10, and 50 mg/kg) in a paddy soil. Real-time quantitative PCR and high-throughput sequencing were conducted to reveal the microbial mechanism of the nanometer effect. The results showed that, though 0.1–10 mg/kg AgNPs had no significant effects on denitrification rate and N2O emission rate compared to CK and bulk Ag treatments, 50 mg/kg AgNPs significantly stimulated more than 60% increase of denitrification rate and N2O emission rate on the 3rd day (P < 0.05). Real-time quantitative PCR revealed that 50 mg/kg AgNPs significantly decreased the abundance of 16S bacterial rRNA gene, nirS/nirK, cnorB, and nosZ genes, but it did not change the narG gene abundance. The correlation analysis further revealed that the cumulative N2O emission was positively correlated with the ratio of all the five tested denitrifying genes to bacterial 16S rRNA gene (P < 0.05), indicating that the tolerance of narG gene to AgNPs was the key factor of the increase in denitrification in the studied soil. High-throughput sequencing showed that only the 50-mg/kg-AgNP treatment significantly changed the microbial community composition compared to bulk Ag and CK treatments. The response of microbial phylotypes to AgNPs suggested that the most critical bacteria which drove the stimulation of 50 mg/kg AgNPs on N2O emission were Firmicutes and β-proteobacteria, such as Clotridiales, Burkholderiales, and Anaerolineales. This study revealed the effects of AgNPs to denitrification in a paddy ecosystem and could provide a scientific basis for understanding of the environmental and toxicological effects of Ag nanomaterials.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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