Efficiency-corrected PCR quantification for identification of prevalence and load of respiratory disease-causing agents in feedlot cattle

Ian Marsh, Rebecca Barnewall, Thomas Williams, Paul Cusack, Narelle Sales, Francesca Galea, Andrew Szentirmay, Jan M Ruijter, Maurice J B van den Hoff, Jane Quinn

Research output: Other contribution to conferenceAbstract

Abstract

From a veterinary diagnostic perspective, PCR is predominantly performed qualitatively, be it conventional or real-time. However, pathogen load
is becoming increasingly important in disease management. Quantitative PCR aims to measure the nucleic acid concentration of a specific target
based on the quantification cycle (Cq) value, derived from the sample amplification curve, plotted on a standard curve generated from purified
nucleic acid standards. Acceptance of the PCR run is often made using the statistical attributes of the standard curve only whilst ignoring similar
criteria from the samples. In doing so, the impact of the sample is often overlooked or underestimated. At best this approach might best be
described as semi-quantitative. Typically, qPCR assumes assays are 100% efficient, that is, produce a doubling of amplified product each cycle.
It could be argued this is rarely the case in a diagnostic assay.
To achieve a more meaningful quantitative result, we used the Cq value and efficiency derived from the sample amplification curve used in
conjunction with the efficiency value from a single calibrator standard (with a known target concentration), a process known as efficiency
corrected quantitative PCR (EC-qPCR). EC-qPCR was used in a recent study to investigate the agents involved in bovine respiratory disease on
cattle at induction to the feedlot. Bovine respiratory disease (BRD) is the most prevalent disease in feedlot cattle worldwide and considered one
of the most difficult and complicated cattle diseases, largely due to the number of agents involved. BRD is commonly attributed to Bovine
alphaherpesvirus 1 (BoAHV1), Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida and Trueperella pyogenes.
https://www.conftool.com/gq2023/index.php?page=browseSessions&print=yes&doprint=yes&form_session=32&presentations=show 2/2
BRD disease investigation is often complicated by the fact that H. somni, M. haemolytica, P. multocida and T. pyogenes are considered normal
flora of cattle and therefore their presence in the upper airways alone is not necessarily informative with respect to disease status or risk. To get a
better understanding of the relationship between presence, load and disease status, we investigated these agents using EC-qPCR to accurately
determine the prevalence and load in the upper respiratory tract from newly inducted cattle were compared with cattle in the hospital pen of the
feedlot. EC-qPCR results and clinical data were combined to establish profiles to elucidate the combinations of agents and those animals’
experiencing proliferation of the agents verses normal levels. Using technology that can produce individual amplification efficiencies for each
sample and EC-qPCR to investigate, analyse and identify BRD-associated viral and bacterial agents represents a new opportunity for Australian
feedlot systems to manage and treat BRD. EC-qPCR opens the scope for any disease investigation where accurate qPCR results are required.
Original languageEnglish
Publication statusPublished - 22 Mar 2023
Event10th Gene Quantification Symposium - qPCR, dPCR and NGS 2023: #GQ2023 - Technical University of Munich, Munich, Germany
Duration: 20 Mar 202324 Mar 2023
https://www.gene-quantification.de/GQ2023/index.html

Conference

Conference10th Gene Quantification Symposium - qPCR, dPCR and NGS 2023
Country/TerritoryGermany
CityMunich
Period20/03/2324/03/23
OtherThe great international interest in the previous nine Gene Quantification Events from 2004 to 2019 with a constant audience of around 400-500 participants from all over the world motivates repeating the success next year. The 10th Gene Quantification Event will take place from 20-24th March 2023. We plan to hold this symposium on-site here at TUM in-person and live.

We broaden our focus in genomics applications from quantitative RT-PCR, to digital-PCR and the latest Next-Generation Sequencing Technologies as well as the connected integrative Multi-Omics data analysis. This time we also present a Sars-Cov-2 Session, about Sars-Cov-2 Quantification, MIQE, Pitfalls and Solutions, ... and more interesting around Covid-19, e.g. Long- or Post- Covid-19 Biomarkers.

As in previous years, we will offer a 3-day scientific symposium with around 70 talks in two lecture halls. Also around 70-100 posters will be presented in dedicated poster sessions. Parallel to the scientific symposium, an Industrial Exhibition will take place and around 30 international companies will present their latest molecular diagnostics, qPCR, digital-PCR and NGS services, hardware, technologies, and software applications. The symposium is followed by various 1-day and 2-day PCR related Workshops, e.g. qPCR or digital-PCR hands-on workshops, microRNA and qPCR data analysis.

Event location is as usual the central lecture hall complex and the foyer at TUM School of Life Sciences (Technical University of Munich) in Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich are part of the largest Biotech cluster in Europe (BioM) representing more than 250 companies and academic institutions, located close to the Munich airport (MUC) directly in the heart of Bavaria.
Internet address

Grant Number

  • P.PSH.0873

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