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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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...

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Quantitative PCR of T7 Bacteriophage from Biopanning
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BactQuant: an enhanced broad-coverage bacterial quantitative real-time PCR assay.

Cindy M Liu1, Maliha Aziz, Sergey Kachur

  • 1Division of Pathogen Genomics, Translational Genomics Research Institute, 3051 W, Shamrell Blvd,, Suite 106, Flagstaff, AZ 86001, USA.

BMC Microbiology
|April 19, 2012
PubMed
Summary

A new quantitative PCR assay, BactQuant, accurately quantifies bacterial load using 16S rRNA gene sequences. This method offers broad coverage for diverse bacterial species, improving community analysis.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Bioinformatics

Background:

  • Accurate bacterial load quantification is crucial for microbial community analysis.
  • A culture-independent method for broad bacterial detection and quantification is needed.
  • Existing methods lack comprehensive coverage for diverse bacterial species.

Purpose of the Study:

  • To design and validate a broad-coverage quantitative PCR (qPCR) assay for bacterial load estimation.
  • To quantify 16S rRNA gene copy number across a wide range of bacteria.
  • To improve bacterial community analysis through a reliable quantification method.

Main Methods:

  • Analysis of 4,938 aligned 16S rRNA gene sequences from the Greengenes database for assay design.
  • Development of a TaqMan® qPCR assay targeting the V3-V4 region.
  • In silico and laboratory-based validation, including coverage analysis of >670,000 sequences and adherence to MIQE guidelines.

Main Results:

  • The BactQuant assay demonstrated broad coverage: 91% of phyla, 96% of genera, and >80% of species had a perfect match.
  • Amplification efficiencies ranged from 81-120% with R² >0.99 for 106 bacterial species, including those with mismatches.
  • Low inter- and intra-run coefficients of variance (<3% and <16% for Ct and copy number, respectively) indicate assay reliability.

Conclusions:

  • BactQuant provides significantly broader bacterial coverage compared to previous universal quantification assays.
  • The assay's in vitro performance exceeded in silico predictions, confirming its robustness.
  • BactQuant is a valuable tool for accurate bacterial load quantification in diverse samples.