Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays

Achenbach, Jenna E.; Topliff, Christina L.; Vassilev, Ventzislav; Donis, Ruben O.; Eskridge, Kent M.; Kelling, Clayton L.

doi:10.1016/j.jviromet.2004.05.004

Cited by 18 publications

(9 citation statements)

References 16 publications

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“…For qRT-PCR a 500-nM-final concentration for each primer was used. Primers were designed to have a similar amplicon size and similar amplification efficiency as required for the application of the Ct (cycle threshold) method as a relative measure of the concentration of target gene in our samples, where Ct represents the number of cycles required for the fluorescent signal to cross the threshold, thus exceeding background level [25]. β-actin was used as a housekeeping gene.…”

Section: Rna Isolation and Quantitative Rt-pcrmentioning

confidence: 99%

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Bin

Caputi

Bistoletti

et al. 2018

Purinergic Signalling

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Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ectoenzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamolesensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and αsmooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by highperformance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.

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Section: Rna Isolation and Quantitative Rt-pcrmentioning

confidence: 99%

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Bin

Caputi

Bistoletti

et al. 2018

Purinergic Signalling

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“…Samples were also tested for the presence of BoHV-1, BVDV-1 and BPIV-3, using a multiplex real-time PCR 70 and BRSV using a published real-time PCR assay. 71 The results of the analyses for the nasal swabs and tissue samples are shown in Table 1 and Table 2, respectively.…”

Section: Mycoplasma Bovis In Australiamentioning

confidence: 99%

Is Mycoplasma bovis a missing component of the bovine respiratory disease complex in Australia?

Horwood¹,

Schibrowski

Fowler³

et al. 2014

Aust Veterinary J

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“…1,3,7,8 However, at present, some veterinary diagnostic laboratories are not yet equipped to perform real-time PCR routinely. Thus, there is a need for sensitive and rapid BRSV diagnostic methods for routine use on postmortem specimens.…”

Section: Introductionmentioning

confidence: 99%

“…Recently, a real-time RT-PCR assay was developed for BRSV diagnosis and has yielded very good results 1 , 3 , 7 , 8 . However, at present, some veterinary diagnostic laboratories are not yet equipped to perform real-time PCR routinely.…”

Section: Introductionmentioning

confidence: 99%

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

Quinting

Robert

Letellier³

et al. 2007

J VET Diagn Invest

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Abstract. Bovine respiratory syncytial virus (BRSV) is associated with severe respiratory disease in cattle. BRSV infection frequently leads to the death of young infected animals. The presence of BRSV in postmortem specimens is routinely detected using indirect immunofluorescence (IIF). However, this technique requires special equipment and considerable expertise. The present paper describes the development of a 1-step ELISA for rapid (1.5 hours) detection of BRSV antigen in organ homogenates. The performance of the new 1-step ELISA was evaluated using bovine postmortem specimens (n 5 108) in comparison with 3 other BRSV diagnostic techniques: indirect immunofluorescence, the Clearview respiratory syncytial virus (RSV) test, and real-time reverse transcriptase polymerase chain reaction (RT-PCR). The relative sensitivity, specificity, and the kappa coefficient of 1-step ELISA, the Clearview RSV electroimmunoassay (EIA), and IIF were calculated, using real-time RT-PCR as the reference test. The new 1-step ELISA was the most sensitive and specific of the 3 tests. Thus, the new 1-step ELISA is a reliable test for detecting BRSV antigen in organ homogenates.

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Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays

Cited by 18 publications

References 16 publications

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Is Mycoplasma bovis a missing component of the bovine respiratory disease complex in Australia?

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

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