• Journal of Veterinary Clinics
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• v.27 no.5
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• pp.508-513
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• 2010

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.

• Journal of Life Science
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• v.26 no.5
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• pp.620-631
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• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.