Comparison of ‘real-time’ and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR

2006 ◽  
Vol 312 (1-2) ◽  
pp. 40-44 ◽  
Author(s):  
Margaret E. Hoadley ◽  
Stephen J. Hopkins
Keyword(s):  
Rna Interference ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr

scholarly journals Assessment of Helicobacter pylori Gene Expression within Mouse and Human Gastric Mucosae by Real-Time Reverse Transcriptase PCR

2001 ◽  
Vol 69 (8) ◽  
pp. 4759-4766 ◽  
Author(s):  
Bachra Rokbi ◽  
Delphine Seguin ◽  
Bruno Guy ◽  
Véronique Mazarin ◽  
Emmanuel Vidor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
16S Rrna ◽  
Mouse Model ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
H Pylori

ABSTRACT Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA >katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, andkatA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification ofH. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.

scholarly journals Normal breathing releases SARS-CoV-2 into the air

2021 ◽  
Author(s):  
Piero Di Carlo ◽  
Katia Falasca ◽  
Claudio Ucciferri ◽  
Bruna Sinjari ◽  
Eleonora Aruffo ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Pcr Detection ◽  
Viral Rna ◽  
Rt Pcr ◽  
Air Samples ◽  
Reverse Transcriptase Pcr ◽  
Normal Breathing ◽  
Exhaled Air ◽  
Negative Contagion

This study tests the release of SARS-CoV-2 RNA into the air during normal breathing, without any sign of possible risk of contagion such as coughing, sneezing or talking. Five patients underwent oropharyngeal, nasopharyngeal and salivary swabs for real-time reverse transcriptase PCR (RT-PCR) detection of SARS-CoV-2 RNA. Direct SARS-CoV-2 release during normal breathing was also investigated by RT-PCR in air samples collected using a microbiological sampler. Viral RNA was detected in air at 1 cm from the mouth of patients whose oropharyngeal, nasopharyngeal and salivary swabs tested positive for SARS-CoV-2 RNA. In contrast, the viral RNA was not identified in the exhaled air from patients with oropharyngeal, nasopharyngeal and salivary swabs that tested negative. Contagion of SARS-CoV-2 is possible by being very close to the mouth of someone who is infected, asymptomatic and simply breathing.

scholarly journals Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

2015 ◽  
Vol 53 (8) ◽  
pp. 2641-2647 ◽  
Author(s):  
Todd N. Wylie ◽  
Kristine M. Wylie ◽  
Richard S. Buller ◽  
Maria Cannella ◽  
Gregory A. Storch
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Limit Of Detection ◽  
Respiratory Illness ◽  
The United States ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr ◽  
Enterovirus D68

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

Comparison of Reverse Transcriptase PCR, Reverse Transcriptase Loop-Mediated Isothermal Amplification, and Culture-Based Assays for Salmonella Detection from Pork Processing Environments

2011 ◽  
Vol 74 (2) ◽  
pp. 294-301 ◽  
Author(s):  
CHAYAPA TECHATHUVANAN ◽  
FRANCES ANN DRAUGHON ◽  
DORIS HELEN D'SOUZA
Keyword(s):  
Reverse Transcriptase ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Water Bath ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Loop Mediated Isothermal Amplification

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37°C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62°C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I–based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry.

scholarly journals Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

2008 ◽  
Vol 74 (13) ◽  
pp. 4226-4230 ◽  
Author(s):  
YoungBin Park ◽  
You-Hee Cho ◽  
YoungMee Jee ◽  
GwangPyo Ko
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Food Borne ◽  
Contaminated Food ◽  
Microbial Contaminants ◽  
Pcr Assays ◽  
Sensitive Method

ABSTRACT We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

Real-Time Reverse Transcriptase PCR for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

2010 ◽  
Vol 73 (3) ◽  
pp. 507-514 ◽  
Author(s):  
CHAYAPA TECHATHUVANAN ◽  
FRANCES ANN DRAUGHON ◽  
DORIS HELEN D'SOUZA
Keyword(s):  
Reverse Transcriptase ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Traditional Methods ◽  
Selective Enrichment ◽  
Reverse Transcriptase Pcr

Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 108 to 100 CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37°C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (Tm) analysis to determine specific Salmonella invA (Tm = 87.5°C) and IAC (Tm = 82°C) products. Improved Salmonella detection up to 101 CFU/25 g of pork and 100 CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 106 CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take ≥1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.

scholarly journals Quantification of CCR5 mRNA in Human Lymphocytes and Macrophages by Real-Time Reverse Transcriptase PCR Assay

2003 ◽  
Vol 10 (6) ◽  
pp. 1123-1128 ◽  
Author(s):  
Jian-Ping Lai ◽  
Ji-Hong Yang ◽  
Steven D. Douglas ◽  
Xu Wang ◽  
Eric Riedel ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Immune Cells ◽  
Dynamic Range ◽  
Human Lymphocytes ◽  
Human Monocyte ◽  
Peripheral Blood Lymphocytes ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr

ABSTRACT CCR5, a β-chemokine receptor, plays an important role in human immunodeficiency virus (HIV) infection of human immune cells, as it is a primary coreceptor for HIV entry into macrophages. We have applied a newly developed real-time reverse transcriptase PCR (RT-PCR) assay for the quantification of CCR5 mRNA in human blood immune cells. The CCR5 real-time RT-PCR assay has a sensitivity of 100 mRNA copies, with a dynamic range of detection between 102 and 106 copies of the CCR5 mRNA transcripts per reaction. The assay is highly reproducible, with an intra-assay coefficient of variation of the threshold cycle of less than 2.07%. When used for quantification of CCR5 mRNA in human monocyte-derived macrophages (MDM) and peripheral blood lymphocytes (PBL), the assay has precision and reproducibility. MDM expressed higher levels of CCR5 mRNA than did PBL. Thus, this assay has the potential and a wide application for the investigation of the role of CCR5 in inflammatory diseases and viral infections, including HIV disease.

scholarly journals Multiplex Quantitative Real-Time Reverse Transcriptase PCR for F+-Specific RNA Coliphages: a Method for Use in Microbial Source Tracking

2006 ◽  
Vol 73 (3) ◽  
pp. 808-814 ◽  
Author(s):  
Marek Kirs ◽  
David C. Smith
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Microbial Contamination ◽  
Accurate Determination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rt Pcr ◽  
The Real ◽  
Reverse Transcriptase Pcr ◽  
Remediation Strategies

ABSTRACT It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F+-specific RNA coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F+-specific RNA coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F+-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.

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