Abstract
Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5’UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.
Highlights
Classical swine fever (CSF) is a highly contagious disease affecting swine, resulting in severe economic losses [1]
We developed reverse transcription loopmediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) determined by hydroxynaphthol blue (HNB) dye-mediated visualization using the naked eye
The 2 μL aliquots of RT-LAMP products were UV visualized by 1% of agarose gel electrophoresis after stained with ethidium bromide
Summary
Classical swine fever (CSF) is a highly contagious disease affecting swine, resulting in severe economic losses [1]. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used as tool for amplification and has emerged as a powerful gene amplification tool due to its simplicity, speed, specificity and costeffectiveness This technique is being used increasingly for rapid detection and typing of emerging viruses such as Severe acute respiratory syndrome coronavirus [17], Japanese encephalitis virus [18], Pseudorabies virus [19] and Classical swine fever virus [20]. Rapid and costeffective RT-LAMP assays for the pre-clinical detection of CSFV visualized directly with the naked eye by addition of SYBR Green have been described [20,21,22]. The high sensitivity and specificity of the RT-LAMP reaction were due to continuous amplification under isothermal conditions This assay employed six primers that recognized eight distinct regions of the 5’UTR gene from CSFV
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