scholarly journals A New Specific and Sensitive RT-qPCR Method Based on Splinted 5′ Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs

2021 ◽  
Vol 7 (3) ◽  
pp. 59
Author(s):  
Marine Lambert ◽  
Abderrahim Benmoussa ◽  
Patrick Provost
Keyword(s):  
Ribosomal Rna ◽  
Quantitative Polymerase Chain Reaction ◽  
Quantitative Detection ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
New Family ◽  
Short Rnas ◽  
Optimized Protocol ◽  
Qpcr Method ◽  
Polymerase Chain

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.

scholarly journals Identification of Abundant and Functional dodecaRNAs (doRNAs) Derived from Ribosomal RNA

2021 ◽  
Vol 22 (18) ◽  
pp. 9757 ◽  
Author(s):  
Marine Lambert ◽  
Abderrahim Benmoussa ◽  
Idrissa Diallo ◽  
Katheryn Ouellet-Boutin ◽  
Véronique Dorval ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Detection Method ◽  
Control Cell ◽  
Annexin Ii ◽  
Quantitative Detection ◽  
Rna Seq ◽  
Argonaute 2 ◽  
New Family ◽  
Short Rnas ◽  
Heterogeneous Nuclear Ribonucleoproteins

Using a modified RNA-sequencing (RNA-seq) approach, we discovered a new family of unusually short RNAs mapping to ribosomal RNA 5.8S, which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. Using a new quantitative detection method that we developed, we confirmed our RNA-seq data and determined that the minimal core doRNA sequence and its 13-nt variant C-doRNA (doRNA with a 5′ Cytosine) are the two most abundant doRNAs, which, together, may outnumber microRNAs. The C-doRNA/doRNA ratio is stable within species but differed between species. doRNA and C-doRNA are mainly cytoplasmic and interact with heterogeneous nuclear ribonucleoproteins (hnRNP) A0, A1 and A2B1, but not Argonaute 2. Reporter gene activity assays suggest that C-doRNA may function as a regulator of Annexin II receptor (AXIIR) expression. doRNAs are differentially expressed in prostate cancer cells/tissues and may control cell migration. These findings suggest that unusually short RNAs may be more abundant and important than previously thought.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2187-2193 ◽  
Author(s):  
Felicia J. Setiono ◽  
Debotri Chatterjee ◽  
Marc Fuchs ◽  
Keith L. Perry ◽  
Jeremy R. Thompson
Keyword(s):  
Polymerase Chain Reaction ◽  
Seasonal Variations ◽  
Quantitative Polymerase Chain Reaction ◽  
Virus Titer ◽  
Tissue Type ◽  
Chain Reaction ◽  
Variable Distribution ◽  
Qpcr Method ◽  
Polymerase Chain ◽  
Virus Distribution

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

Comparison of culture and qPCR methods in detection of mycobacteria from drinking waters

2013 ◽  
Vol 59 (4) ◽  
pp. 280-286 ◽  
Author(s):  
Noora H.J. Räsänen ◽  
Helena Rintala ◽  
Ilkka T. Miettinen ◽  
Eila Torvinen
Keyword(s):  
Surface Water ◽  
Quantitative Polymerase Chain Reaction ◽  
Water Source ◽  
Assimilable Organic Carbon ◽  
Environmental Mycobacteria ◽  
Drinking Waters ◽  
Qpcr Method ◽  
Sensitive Tool ◽  
Different Types ◽  
Polymerase Chain

Environmental mycobacteria are common bacteria in man-made water systems and may cause infections and hypersensitivity pneumonitis via exposure to water. We compared a generally used cultivation method and a quantitative polymerase chain reaction (qPCR) method to detect mycobacteria in 3 types of drinking waters: surface water, ozone-treated surface water, and groundwater. There was a correlation between the numbers of mycobacteria obtained by cultivation and qPCR methods, but the ratio of the counts obtained by the 2 methods varied among the types of water. The qPCR counts in the drinking waters produced from surface or groundwater were 5 to 34 times higher than culturable counts. In ozone-treated surface waters, both methods gave similar counts. The ozone-treated drinking waters had the highest concentration of assimilable organic carbon, which may explain the good culturability. In warm tap waters, qPCR gave 43 times higher counts than cultivation, but both qPCR counts and culturable counts were lower than those in the drinking waters collected from the same sites. The TaqMan qPCR method is a rapid and sensitive tool for total quantitation of mycobacteria in different types of clean waters. The raw water source and treatments affect both culturability and total numbers of mycobacteria in drinking waters.

scholarly journals Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

2017 ◽  
Vol 75 (11) ◽  
pp. 2615-2621 ◽  
Author(s):  
P. Gyawali ◽  
J. P. S. Sidhu ◽  
W. Ahmed ◽  
P. Jagals ◽  
S. Toze
Keyword(s):  
Environmental Samples ◽  
Quantitative Measurement ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Methods ◽  
Quantitative Detection ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Vital Stain ◽  
Polymerase Chain ◽  
Hookworm Ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

Midturbinate Swabs Are Comparable to Nasopharyngeal Swabs for Quantitative Detection of Respiratory Syncytial Virus in Infants

2018 ◽  
Vol 8 (6) ◽  
pp. 554-558 ◽  
Author(s):  
Anne J Blaschke ◽  
Matt McKevitt ◽  
Krow Ampofo ◽  
Tammi Lewis ◽  
Hao Chai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Respiratory Syncytial Virus ◽  
Real Time ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Viral Loads ◽  
Polymerase Chain ◽  
Syncytial Virus

Abstract Nasopharyngeal (NP) swabs are generally used to detect respiratory syncytial virus (RSV) in infants. However, midturbinate (MT) swabs may provide comparable results. In this study, we enrolled hospitalized infants aged <24 months with RSV and collected NP and MT swabs. The resulting viral loads measured by real-time reverse-transcription quantitative polymerase chain reaction were similar. Most parents preferred MT swabs over NP swabs.

scholarly journals Enumeration of viable and non-viable larvated Ascaris eggs with quantitative PCR

2012 ◽  
Vol 10 (4) ◽  
pp. 594-604 ◽  
Author(s):  
Maria Raynal ◽  
Eric N. Villegas ◽  
Kara L. Nelson
Keyword(s):  
Polymerase Chain Reaction ◽  
Detection Limit ◽  
Quantitative Polymerase Chain Reaction ◽  
High Heat ◽  
Chain Reaction ◽  
Direct Microscopy ◽  
Development Stages ◽  
Target Dna ◽  
Qpcr Method ◽  
Polymerase Chain

The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH3-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

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