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. 2023 Sep;35(5):521-527.
doi: 10.1177/10406387231182102. Epub 2023 Jun 20.

Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids

Betsy Armenta-Leyva  1 , Berenice Munguía-Ramírez  1 , Luis G Giménez-Lirola  1 , Xue Lin  1 , Fangshu Ye  2 , Jeffrey Zimmerman  1
Affiliations

Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids

Betsy Armenta-Leyva et al. J Vet Diagn Invest. 2023 Sep.

Abstract

Based on publications reporting improvements in real-time PCR (rtPCR) performance, we compared protocols based on heat treatment or dilution followed by direct rtPCR to standard extraction and amplification methods 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 (OFs). In part A, we subjected aliquots of positive OF samples to 1 of 4 protocols: protocol 1: heat (95°C × 30 min) followed by direct rtPCR; protocol 2: heat and cool (25°C × 20 min) followed by direct rtPCR; protocol 3: heat, cool, extraction, and rtPCR; protocol 4 (control): extraction and then rtPCR. In part B, positive OF samples were split into 3, diluted (D1 = 1:2 with Tris-borate-EDTA (TBE); D2 = 1:2 with negative OF; D3 = not diluted), and then tested by rtPCR using the best-performing protocol from part A (protocol 4). In part A, with occasional exceptions, heat treatment resulted in marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In part B, sample dilution with TBE or OF produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided superior detection of PRRSV, IAV, PEDV, and MHP nucleic acids in OFs.

Keywords: Mycoplasma hyopneumoniae; direct real-time PCR; heat treatment; influenza A virus; oral fluid; porcine epidemic diarrhea virus; porcine reproductive and respiratory syndrome virus; sample dilution.

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Conflict of interest statement

The authors declared no conflicts of interest with respect to their authorship and/or the publication of this manuscript, with the exception that Jeffrey Zimmerman serves as a consultant to Idexx Laboratories. The terms of the consulting arrangement have been reviewed and approved by Iowa State University in accordance with its conflict of interest policies.

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