PMID- 28213086
OWN - NLM
STAT- MEDLINE
DCOM- 20171002
LR  - 20220408
IS  - 1879-0984 (Electronic)
IS  - 0166-0934 (Linking)
VI  - 243
DP  - 2017 May
TI  - Comparison of protocols for the analysis of type 1 porcine reproductive and 
      respiratory syndrome virus by RT-PCR using oral fluids.
PG  - 190-195
LID - S0166-0934(16)30592-4 [pii]
LID - 10.1016/j.jviromet.2017.02.010 [doi]
AB  - The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in 
      oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is 
      gaining increasing popularity. However, the different steps leading to a result 
      have not been extensively evaluated. The aim of the present study was to examine 
      the effect on the performance of qRT-PCR with different sampling materials, 
      conditions of storage of the OF, the need for centrifuging OF, as well as to 
      compare RNA extraction methods and PCR mixes. For the assays, pen-based oral 
      fluids were used, which were pooled and spiked in a serial dilution (up to 
      genotype 10(0) TCID(50)/mL) of type 1 PRRSV isolate 3267. Centrifugation at 
      15,000g for 15min resulted in an increase in sensitivity (1-2 PCR cycles) that 
      was significant (P<0.05) at the lowest dilution tested. The TRIzol and MagMAX RNA 
      extraction methods gave the maximum sensitivity, lowest threshold cycle (Ct), at 
      equivalent virus concentrations. The AgPath-ID One-Step RT-PCR Kit PCR mix 
      reagents were more sensitive for the detection of PRRSV using a purified plasmid 
      as standard, but LSI VetMAX PRRSV EU/NA PRRSV reagents resulted in a slightly 
      better sensitivity with OF (p<0.05). The present results may be useful to 
      standardize protocols for optimizing detection of type 1 PRRSV in OF by qRT-PCR.
CI  - Copyright (c) 2017 Elsevier B.V. All rights reserved.
FAU - Gibert, Elisa
AU  - Gibert E
AD  - Centre de Recerca en Sanitat Animal (CReSA), IRTA-UAB, Edifici CReSA, Campus UAB, 
      08193, Bellaterra, Spain. Electronic address: elisa.gibert@irta.cat.
FAU - Martin-Valls, Gerard
AU  - Martin-Valls G
AD  - Departament de Sanitat i Anatomia Animals, Universitat Autonoma de Barcelona 
      (UAB), Campus UAB, 08193, Bellaterra, Spain. Electronic address: 
      gerard.martin@uab.cat.
FAU - Mateu, Enric
AU  - Mateu E
AD  - Centre de Recerca en Sanitat Animal (CReSA), IRTA-UAB, Edifici CReSA, Campus UAB, 
      08193, Bellaterra, Spain; Departament de Sanitat i Anatomia Animals, Universitat 
      Autonoma de Barcelona (UAB), Campus UAB, 08193, Bellaterra, Spain. Electronic 
      address: enric.mateu@uab.cat.
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20170216
PL  - Netherlands
TA  - J Virol Methods
JT  - Journal of virological methods
JID - 8005839
SB  - IM
MH  - Animals
MH  - *Genotype
MH  - Molecular Diagnostic Techniques/*methods
MH  - Mouth/*virology
MH  - Porcine Reproductive and Respiratory Syndrome/*diagnosis/virology
MH  - Porcine respiratory and reproductive syndrome 
      virus/classification/genetics/*isolation & purification
MH  - Real-Time Polymerase Chain Reaction/*methods
MH  - Sensitivity and Specificity
MH  - Specimen Handling/methods
MH  - Swine
OTO - NOTNLM
OT  - Diagnosis
OT  - Oral fluids
OT  - Porcine reproductive and respiratory syndrome virus
OT  - RNA extraction
OT  - qRT-PCR
EDAT- 2017/02/19 06:00
MHDA- 2017/10/03 06:00
CRDT- 2017/02/19 06:00
PHST- 2016/10/24 00:00 [received]
PHST- 2017/01/12 00:00 [revised]
PHST- 2017/02/13 00:00 [accepted]
PHST- 2017/02/19 06:00 [pubmed]
PHST- 2017/10/03 06:00 [medline]
PHST- 2017/02/19 06:00 [entrez]
AID - S0166-0934(16)30592-4 [pii]
AID - 10.1016/j.jviromet.2017.02.010 [doi]
PST - ppublish
SO  - J Virol Methods. 2017 May;243:190-195. doi: 10.1016/j.jviromet.2017.02.010. Epub 
      2017 Feb 16.