Assessment of strategies to improve routine PCR diagnostics for swine pathogens
Date
2022-12
Authors
Armenta Leyva, Betsy
Major Professor
Advisor
Zimmerman, Jeffrey J
Giménez-Lirola, Luis G
Gauger, Phillip C
Committee Member
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Abstract
There are a vast variety of swine pathogens that can be detected and monitored with PCR technology. Still, to keep up with innovations and improve reproducibility of the technique, this research focused on analytical and technical strategies that were reported to improve PCR applications. Within this context, the objective of this thesis was to assess technical and analytical strategies to improve real-time PCR (qPCR) surveillance of swine pathogens.
The thesis begins with a study focused on normalization of qPCR results. In Chapter 2, a normalization method that converts Cqs to “efficiency standardized” Cqs (ECqs) relative to a reference standard was adapted to a commercial PRRSV RT-qPCR. In this study, matrix-specific reference standards were created by rehydrating and then diluting a PRRSV modified-live virus (MLV) vaccine (Ingelvac® PRRS MLV) with serum or oral fluid. Reference standards were run on each plate and used to calculate ECqs as E-ΔCq, where E is amplification efficiency and ΔCq is the difference between the sample and reference standard Cqs. Serum and oral fluid samples collected from individual pigs vaccinated with a PRRSV MLV (Ingelvac® PRRS MLV) from -7 to 42 days post vaccination were tested and the raw Cqs converted to ECqs to be then analyzed using the receiver operating characteristic analysis. Analysis of the results showed that re-expression of Cqs as ECqs achieved a normalized qPCR response and can be used for evaluation of diagnostic performance and generation of statistically valid cutoffs. Thus, normalization using an agreed-upon reference standard would increase repeatability, reproducibility, and precision in target concentration estimates by accounting for amplification efficiency.
In Chapter 3, sample treatments (heat or dilution) followed by direct qPCR were compared to standard extraction and amplification for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV), or Mycoplasma hyopneumoniae (MHP) in swine oral fluids based on reported improvements in qPCR performance. In Part A, aliquots of positive oral fluid samples were subjected to one of 4 protocols: (Protocol 1) heat (95°C × 30 m) followed by direct qPCR; (Protocol 2) heat and cool (25°C × 20 m) followed by direct qPCR; (Protocol 3) heat, cool, extraction, and qPCR; (Protocol 4, control) extraction and then qPCR. In Part B, positive oral fluid samples were split into three and (D1) diluted 1:2 with TBE; (D2) diluted 1:2 with negative oral fluid; or (D3) not diluted and then qPCR tested using the best performing protocol from Part A. Results in Part A showed that, with occasional exceptions, heat treatment resulted in a marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In Part B, sample dilution with TBE or oral fluid produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided the best results for the detection of PRRSV, IAV, PEDV, and MHP nucleic acids in oral fluids.
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thesis