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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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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Normalizing real-time PCR results in routine testing.

Betsy Armenta-Leyva1, Berenice Munguía-Ramírez1, Ting-Yu Cheng2

  • 1Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Lloyd Veterinary Medical Center, Iowa State University, Ames, IA, USA.

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
|November 3, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces efficiency standardized Cqs (ECqs) for normalizing real-time PCR assays, improving porcine reproductive and respiratory syndrome virus (PRRSV) detection in serum and oral fluid samples. The ECq method enhances diagnostic accuracy for PRRSV surveillance.

Keywords:
ECqamplification efficiencynormalizationreal-time PCRrun-to-run variation

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Area of Science:

  • Veterinary Diagnostics
  • Molecular Biology
  • Virology

Background:

  • Real-time PCR assays require normalization to control for variations in sampling and testing.
  • Quantification cycles (Cqs) in real-time PCR can be converted to efficiency standardized Cqs (ECqs) for improved accuracy.
  • Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to swine health and the economy.

Purpose of the Study:

  • To develop and validate an ECq method for normalizing a commercial PRRSV RT-qPCR assay.
  • To assess the diagnostic performance of the ECq method in serum and oral fluid samples.
  • To improve the accuracy and reliability of PRRSV detection in swine.

Main Methods:

  • ECqs were calculated using the formula E-ΔCq, where E is amplification efficiency and ΔCq is the difference between sample and reference standard Cqs.
  • Reference standards were created using a PRRSV modified-live vaccine diluted in serum or oral fluid.
  • Serum and oral fluid samples from vaccinated pigs were tested, and Cqs were converted to ECqs.

Main Results:

  • The ECq method demonstrated high diagnostic accuracy, with an area under the curve of 0.999 for serum and 0.947 for oral fluid.
  • Diagnostic sensitivity and specificity were high for both sample types, with serum showing 97.9% sensitivity and 100% specificity, and oral fluid showing 82.6% sensitivity and 100% specificity.
  • The amplification efficiency (E) ranged from 1.7 to 2.6 for serum and 1.7 to 2.3 for oral fluid.

Conclusions:

  • The ECq method provides a robust approach for normalizing PRRSV RT-qPCR assays.
  • This normalization strategy enhances the diagnostic sensitivity and specificity of PRRSV detection in both serum and oral fluid.
  • The ECq method holds promise for improved PRRSV surveillance and disease management in swine populations.