Basic principles of real-time quantitative PCR

Arya, Manit; Shergill, Iqbal; Williamson, Magali; Gommersall, Lyndon; Arya, Neehar; Patel, Hitendra

doi:10.1586/14737159.5.2.209

Cited by 481 publications

(361 citation statements)

References 66 publications

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“…Relative mRNA levels were determined using the Bio-Rad iCycler and SYBR Green method (Invitrogen; S7563) [43]. The following primers were used: IL-1b forward (5 0 -TGGCCCAGG-CGTCAGA-3 0 ), IL-1b reverse (5 0 -GGTTTGCTACAACATGGGCT-ACA-3 0 ); TNF-a forward (5 0 -GCCCTAAACAGATGAAGTG-CTC-3 0 ), TNF-a reverse (5 0 -GAACCAGCATCTTCCTCAG-3 0 ); B2M forward (5 0 -ATGAGTATGCCTGCCGTGTG-3 0 ), B2M reverse (5 0 -CCAAATGCGGCATCTTCAAAC-3 0 ) (Biolegio, Malden, The Netherlands).…”

Section: Cytokine Measurementsmentioning

confidence: 99%

Engagement of NOD2 has a dual effect on proIL‐1β mRNA transcription and secretion of bioactive IL‐1β

et al. 2007

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Synthesis and release of pro-inflammatory cytokines, such as IL-1b, play a crucial role in the intestinal inflammation that characterizes Crohn's disease. Mutations in the nucleotide oligomerization domain 2 (NOD2) gene are associated with an increased risk of Crohn's disease. Although it is known that NOD2 mediates cytokine responses to muramyl dipeptide (MDP), it is yet unclear whether NOD2 stimulation mediates only transcription of pro-IL-1b mRNA, or whether NOD2 is also involved in the activation of caspase-1 and release of active IL-1b. By investigating the response of MNC from Crohn's disease patients homozygous for the 3020insC NOD2 mutation, we were able to show that NOD2 signaling after stimulation with MDP has a dual effect by activating proIL-1b mRNA transcription and inducing release of bioactive IL-1b. Because NOD2 engagement amplifies TLR stimulation, we investigated whether activation of caspase-1 by MDP is involved in the NOD2/TLR synergism. The synergy in IL-1b production between NOD2 and TLR is mediated at post-translational level in a caspase-1-dependent manner, which indirectly suggests that NOD2 also induces caspase-1 activation. In contrast, the synergy in TNF-a production after stimulation with MDP and LPS is induced at transcriptional level. This demonstrates that both caspase-1-dependent and -independent mechanisms are involved in the synergy between NOD2 and TLR.Introduction NOD-like receptors (NLR) are intracellular receptors for bacterial peptidoglycans, which complement the recognition of pathogen-associated molecular patterns (PAMP) by membrane-bound TLR [1,2]. Nucleotide oligomerization domain 2 (NOD2) is a member of the NACHT-LRR (NLR) receptor family, which recognizes muramyl dipeptide (MDP), the minimal motif of peptidoglycan of both Gram-positive and Gram-negative bacteria [3]. Mutations in the NOD2 gene are associated with Crohn's disease [4,5], but how NOD2 exactly acts in the pathogenesis of this auto-inflammatory disease is unclear [6][7][8]. Therefore, a better understanding of the intracellular events induced by the interaction between NOD2 and peptidoglycan is crucial for both the insight into recognition of Gram-positive pathogens by the innate immune system, and for the pathogenesis of the inflammatory reactions in Crohn's disease. Activation of human mononuclear cells (MNC) by MDP leads to production of pro-inflammatory cytokines, especially IL-1b [7,9]. IL-1b is produced as pro-IL-1b a 31-34-kDa inactive form of the cytokine, which is later cleaved by caspase-1 to the bioactive 17-kDa . This is followed by IL-1b excretion in microvesicles into the extracellular environment [11]. Apparently, MDP is capable of inducing all three steps, but it is unclear whether NOD2 alone or other receptors are involved in one or more of these steps of IL-1b production. It has been proposed that several of the NLR family members are able to recognize MDP, most notably NOD2, NALP3, and NALP1, and that they execute different functions necessary for cytokine production. In this concept, ...

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Section: Cytokine Measurementsmentioning

confidence: 99%

Engagement of NOD2 has a dual effect on proIL‐1β mRNA transcription and secretion of bioactive IL‐1β

et al. 2007

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“…As soon as annealing between the primer-probe and the target occurs, scorpion primer combines to the PCR product and then the probe sequence in the tail curls back to hybridise with the sequence of target. As the tail of the scorpion and the amplicon are part of the same strand of DNA, the interaction is intra-molecular (Giulietti et al 2001;Arya et al 2005). Hybridisation reaction unties the hairpin loop, separating the fluorophore and the quencher, which leads to an increase in the fluorescence emitted (Figure 4).…”

Section: Detection Based On Sequence Specific Methodsmentioning

confidence: 99%

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

Mirmajlessi¹,

Loit²,

Mänd³

et al. 2015

Plant Prot. Sci.

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Mirmajlessi S.M., Loit E., Mänd M., Mansouripour S.M. (2015): Real-time PCR applied to study on plant pathogens: potential applications in diagnosis -a review. Plant Protect Sci., 51: 177-190.Quantitative real-time PCR (qPCR) technique incorporates traditional polymerase chain reaction (PCR) efficiency with the production of a specific fluorescent signal, measuring the kinetics of the reaction in the early PCR phases and providing quantification of specific targets in various environmental samples. There are an increasing number of chemistries to detect PCR products, which are widely used in plant pathology as they cluster into the amplicon sequence non-specific and sequence-specific techniques. In this review, we illustrate a general description of major chemistries and discuss some considerations for assay development as it applies for a wide range of applications in epidemiological studies. The technique has become the gold standard for early detection of pathogens and a fundamental tool in the research laboratory.

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“…Because of the low adaptation to cell culture, it is difficult to differentiate PEDV from other enteric viral pathogens using cell culture isolation (16). In addition, due to the high frequency of mutations, various PEDV epidemic strains cannot be correctly identified using conventional primers for PCR assay (1). Real-time RT-PCR with specific primer pair and probe is a more specific and sensitive technique for the identification of the presence of the virus in field samples, and also provides the capacity to quantify the virus (15).…”

Section: Introductionmentioning

confidence: 99%

Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

Hou

Xie

Wang

et al. 2016

Journal of Veterinary Research

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Introduction: A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed. Material and Methods: Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999. Results: The developed quantitative PCR assay detected viral titres as low as 0.1 TCID50 with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive. Conclusion: This approach is suitable for clinical sample identification and pathogenesis studies.

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Basic principles of real-time quantitative PCR

Cited by 481 publications

References 66 publications

Engagement of NOD2 has a dual effect on proIL‐1β mRNA transcription and secretion of bioactive IL‐1β

Engagement of NOD2 has a dual effect on proIL‐1β mRNA transcription and secretion of bioactive IL‐1β

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

Development and application of a TaqMan-MGB real-time RT-PCR assay for the detection of porcine epidemic diarrhoea virus strains in China

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