scholarly journals Simultaneous Absolute Quantification of Target and Control Templates by Real-Time Fluorescence Reverse Transcription-PCR Using 4-(4′-Dimethylaminophenylazo)benzoic Acid as a Dark Quencher Dye

2001 ◽  
Vol 47 (3) ◽  
pp. 486-490 ◽  
Author(s):  
Karl-Anton Kreuzer ◽  
Alexander Bohn ◽  
Joachim Lupberger ◽  
Jerome Solassol ◽  
Philipp le Coutre ◽  
...  
Keyword(s):  
Benzoic Acid ◽  
Real Time ◽  
Correlation Coefficient ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Rt Pcr ◽  
Internal Reference ◽  
Reverse Transcription Pcr

Abstract Background: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. Methods: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 β-actin molecules as control templates, 105 bcr/abl molecules were amplified, and 105 β-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). Results: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10–105 copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for β-actin (coamplified with M-bcr/abl or m-bcr/abl) for 103–107 copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. Conclusions: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and β-actin transcripts allows monitoring of bcr/abl+ leukemias.

scholarly journals Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

2002 ◽  
Vol 68 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
Camile Pizeta Semighini ◽  
Mozart Marins ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Aspergillus Nidulans ◽  
Abc Transporter ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Reverse Transcription Pcr ◽  
Nitroquinoline Oxide

ABSTRACT The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

scholarly journals Circulating Prostate Tumor Cells Detected by Reverse Transcription-PCR in Men with Localized or Castration-Refractory Prostate Cancer: Concordance with CellSearch Assay and Association with Bone Metastases and with Survival

2009 ◽  
Vol 55 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Pauliina Helo ◽  
Angel M Cronin ◽  
Daniel C Danila ◽  
Sven Wenske ◽  
Rita Gonzalez-Espinoza ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Metastases ◽  
Healthy Volunteers ◽  
Real Time ◽  
Tumor Cells ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Reverse transcription-PCR (RT-PCR) assays have been used for analysis of circulating tumor cells (CTCs), but their clinical value has yet to be established. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs via real-time RT-PCR assays for KLK3 [kallikrein-related peptidase 3; i.e., prostate-specific antigen (PSA)] and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival. Methods: KLK3, KLK2, and PSCA (prostate stem cell antigen) mRNAs were measured by standardized, quantitative real-time RT-PCR assays in blood samples from 180 localized-disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™; Veridex) approved for clinical use. Results: All healthy volunteers were negative for KLK mRNAs. Results of tests for KLK3 or KLK2 mRNAs were positive (≥80 mRNAs/mL blood) in 37 patients (49%) with CRPC but in only 15 patients (8%) with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80%–85%) and correlated (Kendall τ, 0.60–0.68). Among CRPC patients, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival but were only modestly correlated with serum PSA concentrations. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with a positive KLK mRNA status. Conclusions: Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases but are rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals An Integrated Diagnostic Approach for Citrus Exocortis Viroid

2021 ◽  
Author(s):  
Chih-Hsu Lin ◽  
Ting-Hsuan Hung ◽  
I Hu ◽  
Ta-Hsin Ku ◽  
Chun-Yi Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Severe Disease ◽  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Cultivar ◽  
Dot Blot Hybridization ◽  
Reverse Transcription Pcr ◽  
One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

scholarly journals Triplex Real Time RT-PCR for N1, N2 and RP probes from CDC EUA SARS-CoV-2 diagnosis kit

2020 ◽  
Author(s):  
Byron Freire-Paspuel ◽  
Patricio Vega-Mariño ◽  
Alberto Velez ◽  
Marilyn Cruz ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Speed Up

AbstractCDC protocol for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 3 targets for detection (N1, N2 and RP) labelled with FAM so 3 PCR reactions are required per sample. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with CDC singleplex assay. This protocol could speed up detection and save reagents during current SARS-CoV-2 testing supplies shortage.

scholarly journals Major Interference from Leukocytes in Reverse Transcription-PCR Identified as Neurotoxin Ribonuclease from Eosinophils: Detection of Residual Chronic Myelogenous Leukemia from Cell Lysates by Use of an Eosinophil-depleted Cell Preparation

1999 ◽  
Vol 45 (4) ◽  
pp. 465-471 ◽  
Author(s):  
Mauri M Hämäläinen ◽  
Jarkko U Eskola ◽  
Jukka Hellman ◽  
Kari Pulkki
Keyword(s):  
Amino Acid ◽  
Chronic Myelogenous Leukemia ◽  
Reverse Transcription ◽  
Mononuclear Cells ◽  
Rna Extraction ◽  
Myelogenous Leukemia ◽  
Rnase Activity ◽  
Rt Pcr ◽  
Cell Lysates ◽  
Reverse Transcription Pcr

Abstract Background: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 105 leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes. Methods: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque® density gradient. Results: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by ∼90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 105 CD16+ granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control. Conclusion: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.

scholarly journals Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

2007 ◽  
Vol 53 (11) ◽  
pp. 1899-1905 ◽  
Author(s):  
Marit Kramski ◽  
Helga Meisel ◽  
Boris Klempa ◽  
Detlev H Krüger ◽  
Georg Pauli ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Sequence Information ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

scholarly journals Complete Genome Sequence of Enterovirus D68 Detected in Classroom Air and on Environmental Surfaces

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
John A. Lednicky ◽  
Tania S. Bonny ◽  
J. Glenn Morris ◽  
Julia C. Loeb
Keyword(s):  
Real Time ◽  
Genome Sequence ◽  
Reverse Transcription ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Sampling Method ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Enterovirus D68

We amplified and sequenced the complete genome of enterovirus D68 (EV-D68) that had been collected from classroom air using a filter-based air sampling method and by swab sampling of environmental surfaces. Relatively high levels of EV-D68 genome equivalents were found per cubic meter of air by quantitative real-time reverse transcription-PCR (RT-PCR).

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