PMID- 37919959
OWN - NLM
STAT- MEDLINE
DCOM- 20231221
LR  - 20231223
IS  - 1943-4936 (Electronic)
IS  - 1040-6387 (Print)
IS  - 1040-6387 (Linking)
VI  - 36
IP  - 1
DP  - 2024 Jan
TI  - Normalizing real-time PCR results in routine testing.
PG  - 78-85
LID - 10.1177/10406387231206080 [doi]
AB  - Normalization, the process of controlling for normal variation in sampling and 
      testing, can be achieved in real-time PCR assays by converting sample 
      quantification cycles (Cqs) to "efficiency standardized Cqs" (ECqs). We 
      calculated ECqs as E(-DeltaCq), where E is amplification efficiency and DeltaCq is the 
      difference between sample and reference standard Cqs. To apply this approach to a 
      commercial porcine reproductive and respiratory syndrome virus (PRRSV) RT-qPCR 
      assay, we created reference standards by rehydrating and then diluting 
      (1 x 10(-4)) a PRRSV modified-live vaccine (PRRS MLV; Ingelvac) with serum or 
      oral fluid (OF) to match the sample matrix to be tested. Sample ECqs were 
      calculated using the mean E and reference standard Cq calculated from the 4 
      reference standards on each plate. Serum (n = 132) and OF (n = 130) samples were 
      collected from each of 12 pigs vaccinated with a PRRSV MLV from -7 to 42 d 
      post-vaccination, tested, and sample Cqs converted to ECqs. Mean plate Es were 
      1.75-2.6 for serum and 1.7-2.3 for OF. Mean plate reference standard Cqs were 
      29.1-31.3 for serum and 29.2-31.5 for OFs. Receiver operating characteristic 
      analysis calculated the area under the curve for serum and OF sample ECqs as 
      0.999 (95% CI: 0.997, 1.000) and 0.947 (0.890, 1.000), respectively. For serum, 
      diagnostic sensitivity and specificity of the commercial PRRSV RT-qPCR assay were 
      estimated as 97.9% and 100% at an ECq cutoff >/= 0.20, and for OF, 82.6% and 100%, 
      respectively, at an ECq cutoff >/= 0.45.
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, Lloyd Veterinary Medical Center, 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, Lloyd Veterinary Medical Center, Iowa State University, 
      Ames, IA, USA.
FAU - Cheng, Ting-Yu
AU  - Cheng TY
AUID- ORCID: 0000-0002-3732-9147
AD  - Department of Veterinary Preventive Medicine, College of Veterinary Medicine, the 
      Ohio State University, Columbus, OH, 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 - Henao-Diaz, Alexandra
AU  - Henao-Diaz A
AD  - Pig Improvement Mexico, Santiago de Queretaro, Queretaro, Mexico.
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, Lloyd Veterinary Medical Center, 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, Lloyd Veterinary Medical Center, Iowa State University, 
      Ames, IA, USA.
LA  - eng
PT  - Journal Article
DEP - 20231102
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
RN  - 0 (Antibodies, Viral)
RN  - 0 (Viral Vaccines)
RN  - 0 (Vaccines, Attenuated)
RN  - O0408QB48D (5-chloroquinoxaline-2-sulfanilamide)
SB  - IM
MH  - Swine
MH  - Animals
MH  - *Porcine Reproductive and Respiratory Syndrome/diagnosis/prevention & control
MH  - Real-Time Polymerase Chain Reaction/veterinary
MH  - Antibodies, Viral
MH  - *Viral Vaccines
MH  - *Porcine respiratory and reproductive syndrome virus/genetics
MH  - Vaccines, Attenuated
MH  - *Swine Diseases/diagnosis
PMC - PMC10734596
OTO - NOTNLM
OT  - ECq
OT  - amplification efficiency
OT  - normalization
OT  - real-time PCR
OT  - run-to-run variation
COIS- Declaration of conflicting interestsThe authors declared no conflicts of interest 
      with respect to their authorship and/or the publication of this manuscript, with 
      the exception that Jeffrey Zimmerman, who 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- 2023/11/03 06:44
MHDA- 2023/12/21 06:42
PMCR- 2024/11/02
CRDT- 2023/11/03 03:41
PHST- 2024/11/02 00:00 [pmc-release]
PHST- 2023/12/21 06:42 [medline]
PHST- 2023/11/03 06:44 [pubmed]
PHST- 2023/11/03 03:41 [entrez]
AID - 10.1177_10406387231206080 [pii]
AID - 10.1177/10406387231206080 [doi]
PST - ppublish
SO  - J Vet Diagn Invest. 2024 Jan;36(1):78-85. doi: 10.1177/10406387231206080. Epub 
      2023 Nov 2.