Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines

Duvigneau, J. Catharina; Hartl, Romana T.; Groiss, Sandra; Gemeiner, Manfred

doi:10.1016/j.jim.2005.06.021

Cited by 128 publications

(66 citation statements)

References 27 publications

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“…Previous studies using real-time RT-PCR to quantify cytokine expression in humans and veterinary species describe the kinetics of cytokine mRNA expression following stimulation, and demonstrate some variation among species (Abdalla et al, 2003;Konnai et al, 2003;Duvigneau et al, 2005;Budhia et al, 2006). Similar to our results with rabbit cells, IFN-γ and IL-2 expression in the cells of humans and other species display a sharp upregulation by 4 hours following mitogen stimulation and peaks between 12-36 hours (Abdalla et al, 2003;Konnai et al, 2003).…”

Section: Discussionsupporting

confidence: 88%

“…Recently, real-time RT-PCR techniques have been developed as a quick, accurate, and relatively inexpensive method for quantitating cytokine responses in human and mouse tissues (Overbergh et al, 1999;Abdalla et al, 2003). Recently, real-time RT-PCR methods have also enabled the study of cytokines in many important veterinary species that, like the rabbit, lack commercially available immunological reagents (Konnai et al, 2003;Duvigneau et al, 2005;Budhia et al, 2006). In the present study, we report the development of a quantitative real time RT-PCR assay for measuring rabbit cytokine expression.…”

Section: Discussionmentioning

confidence: 99%

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Quantitation of rabbit cytokine mRNA by real-time RT-PCR

et al. 2007

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Fundamental understanding of rabbit immunology and the use of the rabbit as a disease model have long been hindered by the lack of immunological assays specific to this species. In the present study, we sought to develop a method to quantitate cytokine expression in rabbit cells and tissues. We report the development of a quantitative real-time RT-PCR method for measuring the relative levels of rabbit IFN-γ, IL-2, IL-4, IL-10 and TNF-α mRNA. Quantitation was accomplished by comparison to a standard curve generated using plasmid DNA containing partial sequences of the relevant cytokines. Experimental studies demonstrate applicability of this assay to quantitate cytokine mRNA levels from rabbit spleen cells following mitogen stimulation. We have further utilized this assay to also examine cytokine expression in rabbit tissues during experimental syphilis infection.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

et al. 2007

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“…As a result of the improvement of cycler platforms and new reporter dyes like NED (575 nm) or Cy5 (667 nm) with distinct emission maxima from, eg, FAM (518 nm) or VIC (554 nm), the development and validation of triplex real-time PCR assays for pathogens or mRNA have been reported recently. [12][13][14] In the present study, a triplex real-time assay for the simultaneous detection of HSV-1, HSV-2, and Internal Control DNA, based on the detection of three different probe-coupled dyes (FAM, NED, and VIC) in a single PCR reaction was evaluated on the ABI Prism 7000. To our knowledge, this assay is the only European Community-approved commercially available kit for HSV-DNA detection and differentiation on the ABI Prism platforms ABI 7900 and 7000 using the triplex PCR technology.…”

Section: Discussionmentioning

confidence: 99%

Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and 2

Reil

Bartlime

Drerup³

et al. 2008

The Journal of Molecular Diagnostics

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A new triplex real-time polymerase chain reaction (PCR) assay for Herpes simplex virus (HSV) (artus HSV-1/2 TM PCR kit , QIAGEN) was evaluated. This assay simultaneously uses three differently labeled probes targeted to HSV-1 (FAM) , HSV-2 (NED) , and to the manufacturer's Internal Control (VIC). HSV-1/2 typing capability and quantitation accuracy were determined using HSV stocks and quality control panels. Performance in routine clinical testing was compared with a nested HSV-1/2 PCR assay. Dilution series and quality control panel testing revealed an approximately 10-fold higher HSV-2 sensitivity in real-time PCR compared with an in-house nested PCR assay. The sensitivity for HSV-1 was comparable in both assays. All HSV-positive proficiency panel samples (n ‫؍‬ 21) and virus stocks were typed correctly as HSV-1 or HSV-2 using real-time PCR. Quantitation correlated well with reference values (HSV-1 , r ‫؍‬ 0.98; HSV-2 , r ‫؍‬ 0.88) , and 95% detection limits were determined as 9.4 HSV-2 copies/reaction and 18 HSV-1 copies/reaction. Based on C t values , the mean intraassay coefficient of variation was 1% , whereas the interassay coefficient of variations were 2.7% and 2.5% for HSV-1 and -2 , respectively. Testing of 309 clinical samples resulted in 100% specificity and 97% sensitivity. In conclusion, the artus HSV-1/2 TM PCR kit represents an excellent tool for the detection and differentiation of HSV-1 and -2 in clinical samples. (J Mol

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“…Reference samples for the different tissues (nasal mucosa, trigeminal ganglion, and CNS tissues) were tested in duplicate before and after preamplification for the different reference TABLE 1 Primers and probes for seven porcine reference genes, eleven cytokines, and six PRV genes were either selected from previous studies or designed using the PrimerQuest tool , gC, gE, and LAT intron) were either selected from previous studies (28)(29)(30)(31) or designed using the PrimerQuest tool (Integrated DNA Technologies [IDT], Leuven, Belgium). Porcine sequences available in the NCBI databases were used to design qPCR assays, taking into account the amplicon specificities of the primers (BLAST).…”

Section: Methodsmentioning

confidence: 99%

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression

Verpoest

Cay

Favoreel

et al. 2017

J Virol

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The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2-and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb.IMPORTANCE It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

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Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines

Cited by 128 publications

References 27 publications

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

Quantitation of rabbit cytokine mRNA by real-time RT-PCR

Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and 2

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression

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