PMID- 37337714
OWN - NLM
STAT- MEDLINE
DCOM- 20230818
LR  - 20230901
IS  - 1943-4936 (Electronic)
IS  - 1040-6387 (Print)
IS  - 1040-6387 (Linking)
VI  - 35
IP  - 5
DP  - 2023 Sep
TI  - Critical evaluation of strategies to achieve direct real-time PCR detection of 
      swine pathogens in oral fluids.
PG  - 521-527
LID - 10.1177/10406387231182102 [doi]
AB  - 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 degrees C x 30 min) 
      followed by direct rtPCR; protocol 2: heat and cool (25 degrees C x 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.
FAU - Armenta-Leyva, Betsy
AU  - Armenta-Leyva B
AUID- ORCID: 0000-0003-4094-6813
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, IA, USA.
FAU - Munguia-Ramirez, Berenice
AU  - Munguia-Ramirez B
AUID- ORCID: 0000-0002-2557-1817
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, IA, USA.
FAU - Gimenez-Lirola, Luis G
AU  - Gimenez-Lirola LG
AUID- ORCID: 0000-0002-4407-7996
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, IA, USA.
FAU - Lin, Xue
AU  - Lin X
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, IA, USA.
FAU - Ye, Fangshu
AU  - Ye F
AUID- ORCID: 0000-0001-6994-1210
AD  - Department of Statistics, College of Liberal Arts and Sciences, Iowa State 
      University, Ames, IA, USA.
FAU - Zimmerman, Jeffrey
AU  - Zimmerman J
AUID- ORCID: 0000-0002-8107-2422
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, IA, USA.
LA  - eng
PT  - Journal Article
DEP - 20230620
PL  - United States
TA  - J Vet Diagn Invest
JT  - Journal of veterinary diagnostic investigation : official publication of the 
      American Association of Veterinary Laboratory Diagnosticians, Inc
JID - 9011490
SB  - IM
MH  - Swine
MH  - Animals
MH  - Real-Time Polymerase Chain Reaction/veterinary/methods
MH  - *Porcine respiratory and reproductive syndrome virus/genetics
MH  - *Swine Diseases/diagnosis
MH  - *Porcine epidemic diarrhea virus
MH  - *Influenza A virus
MH  - *Porcine Reproductive and Respiratory Syndrome/diagnosis
PMC - PMC10467463
OTO - NOTNLM
OT  - Mycoplasma hyopneumoniae
OT  - direct real-time PCR
OT  - heat treatment
OT  - influenza A virus
OT  - oral fluid
OT  - porcine epidemic diarrhea virus
OT  - porcine reproductive and respiratory syndrome virus
OT  - sample dilution
COIS- 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.
EDAT- 2023/06/20 06:41
MHDA- 2023/08/18 06:42
PMCR- 2024/06/20
CRDT- 2023/06/20 03:35
PHST- 2024/06/20 00:00 [pmc-release]
PHST- 2023/08/18 06:42 [medline]
PHST- 2023/06/20 06:41 [pubmed]
PHST- 2023/06/20 03:35 [entrez]
AID - 10.1177_10406387231182102 [pii]
AID - 10.1177/10406387231182102 [doi]
PST - ppublish
SO  - J Vet Diagn Invest. 2023 Sep;35(5):521-527. doi: 10.1177/10406387231182102. Epub 
      2023 Jun 20.