环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种可应用于各种病原体检测、基因分型等领域的等温扩增技术.该技术所需设备简单且易于操作,因此具有较大的发展潜力.根据目前已经存在的判定LAMP结果的可视化方法,以可能对野生小反刍动物造成威胁的小反刍兽疫病毒(Peste des petits rumi-nants virus,PPRV)cDNA为检测对象,选择合适的引物组,比较评估均可在日光或紫外光下区分阴阳性结果的SYBR Green I、SYBR Safe Stain和羟基萘酚蓝(hydroxy naphthol blue,HNB)3种指示剂方法的适用性与灵敏度.结果表明:3种方法的灵敏度都很高,均与琼脂糖凝胶电泳相当.其中,SYBR Green I结果区分度高、不受体系成分影响且无须额外设备,但成本较高;SYBR Safe Stain具有良好的结果区分度,成本相对较低,但需要额外的紫外照射设备;HNB成本最低,但颜色区分度较低,且易受多种因素影响.建议在选择LAMP指示剂时,综合考虑成本、使用便利性、结果准确性及实验条件,通过充分的体系优化和验证,确保LAMP检测的准确性和可靠性.研究结果突显了不同方法的特征和适用场景,为研究人员根据不同场所与条件选择高效、便捷的LAMP反应结果可视化方法提供参考.
A Comparative Study on the Visual Detection Methods of LAMP Reaction Results of PPR
Loop-mediated isothermal amplification(LAMP)is one of the techniques that can be used in detecting and ge-notyping various pathogens,and has significant development potential because of needing simple equipment and ease of operation.According to existing visual detection methods for LAMP,this study focused on detecting the cDNA of PPRV that poses a threat to wild small ruminants.We selected appropriate primers and compared and evaluated the applicability and sensitivity of three indicator methods,SYBR Green I,SYBR Safe Stain,and hydroxy naphthol blue(HNB),which can dif-ferentiate negative and positive results under visible light or ultraviolet light.The results showed that all three methods dem-onstrated high sensitivity,comparable to agarose gel electrophoresis.Among them,SYBR Green Ⅰ shows superior result differentiation,is unaffected by other components,and requires no additional equipment,albeit at higher cost.SYBR Safe Stain provides good result differentiation at a relatively lower cost but necessitates additional UV equipment.HNB is the most cost-effective option but it has lower color differentiation and is susceptible to various influencing factors.It is recom-mended that the selection of LAMP indicators should consider cost,ease of use,result accuracy,and experimental condi-tions.Comprehensive system optimization and validation are advised to ensure the accuracy and reliability of LAMP detec-tion.The characteristics and applicable scenarios of different methods are emphasized,which provided a reference for re-searchers to select efficient and convenient LAMP reaction visualization methods according to different sites and conditions.
万方数据
维普
