Rrt-pcr Detection
Rrt-pcr Detection - Related Videos
12:04
RNA Analysis of Environmental Samples Using RT-PCR
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2023
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona Demonstrating Author: Bradley Schmitz Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies deoxyribonucleic acids (DNA), RT-PCR enables the amplification of ribonucleic acids (RNA) through the formation of complementary DNA (cDNA). This enables RNA-based organisms...
15:16
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
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Cited by 13 •
2012
A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children’s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.
10:55
Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR
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Cited by 28 •
2018
This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.
02:57
Real Time RT-PCR
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2021
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction. The real-time quantification of the number of amplified products is...
12:32
EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR
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Cited by 13 •
2016
Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.
FAQs
Reverse transcription converts the RNA target into complementary DNA before amplification begins. This conversion creates a DNA template that can undergo the subsequent thermal-cycling steps, while preserving the connection to the original RNA signal. The two-stage sequence allows researchers to investigate RNA from viruses or microbes in clinical and experimental infection samples.
Fluorescence provides a signal that increases as amplified product accumulates during thermal cycling. The instrument tracks this change rather than relying only on a final endpoint measurement. Researchers use the cycle threshold, which relates signal detection to amplification progress, to quantify the RNA target and compare infection-related measurements.
Both approaches generate fluorescence as amplification proceeds, but they report the accumulating product differently. Fluorescent dyes produce signal associated with amplified material, whereas sequence-specific probes produce signal when the relevant target sequence is detected. This distinction gives researchers alternative ways to connect amplification with RNA-target measurement.
The cycle threshold provides a measurement point within the amplification process rather than after all cycling is complete. Because fluorescence is monitored as product accumulates, this threshold connects the observed signal with the amount of RNA being measured. It therefore supports quantitative interpretation of pathogen detection and infection-related samples.
A typical workflow begins with an RNA-containing clinical or experimental sample. Reverse transcriptase first produces complementary DNA, and thermal cycling then amplifies the target sequence. Fluorescent dyes or sequence-specific probes generate signals during amplification, after which the cycle threshold is used to interpret the resulting RNA measurement.
Researchers apply the method when they need to identify viral or microbial RNA in clinical or experimental samples. Beyond indicating whether a target is present, the assay can support pathogen identification and estimation of pathogen load. These measurements help characterize infection dynamics and evaluate changes associated with treatment.
In this research context, RNA measurements provide a way to follow the presence and quantity of infectious targets while an infection develops or responds to treatment. Detecting viral or microbial RNA in relevant samples can connect pathogen measurements with experimental observations, helping investigators monitor infection dynamics and treatment-associated responses.