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

Armenta-Leyva, Betsy; Munguía-Ramírez, Berenice; Giménez-Lirola, Luis; Lin, Xue; Ye, Fangshu; Zimmerman, Jeffrey

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

File

2023-Gimenez-Lirola-CriticalEvaluation.pdf (251.42 KB)

Date

2023-06-20

Authors

Armenta-Leyva, Betsy

Munguía-Ramírez, Berenice

Giménez-Lirola, Luis

Lin, Xue

Ye, Fangshu

Zimmerman, Jeffrey

Publisher

Sage Journals

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.

Academic or Administrative Unit

Veterinary Diagnostic and Production Animal Medicine

Type

article

Comments

This is a manuscript of an article published as Armenta-Leyva, Betsy, Berenice Munguía-Ramírez, Luis G. Giménez-Lirola, Xue Lin, Fangshu Ye, and Jeffrey Zimmerman. "Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids." Journal of Veterinary Diagnostic Investigation (2023): 10406387231182102. doi: https://doi.org/10.1177/10406387231182102. Posted with Permission. Copyright the Authors 2023.