RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

作者全名："Zhang, Zhang; Guan, Luhao; Yao, Juan; Li, Lijia; Liu, Chunfang; Guo, Yongcan; Xie, Guoming"

作者地址："[Zhang, Zhang; Guan, Luhao; Xie, Guoming] Chongqing Med Univ, Key Lab Clin Lab Diagnost, Chinese Minist Educ, Coll Lab Med,Chongqing Med Lab Microfluid & SPRi E, Chongqing 400016, Peoples R China; [Guan, Luhao] Luzhou Tradit Chinese Med Hosp, Dept Lab Med, Luzhou 646000, Peoples R China; [Yao, Juan; Guo, Yongcan] Southwest Med Univ, Dept Lab Med, Affiliated Tradit Chinese Med Hosp, Luzhou 646000, Peoples R China; [Li, Lijia; Liu, Chunfang] Zhuhai Biori Biotechnol Co Ltd, Zhuhai 519000, Peoples R China"

通信作者："Zhang, Z; Xie, GM (通讯作者)，Chongqing Med Univ, Key Lab Clin Lab Diagnost, Chinese Minist Educ, Coll Lab Med,Chongqing Med Lab Microfluid & SPRi E, Chongqing 400016, Peoples R China.; Guo, YC (通讯作者)，Southwest Med Univ, Dept Lab Med, Affiliated Tradit Chinese Med Hosp, Luzhou 646000, Peoples R China."

来源：ANALYTICAL CHEMISTRY

ESI学科分类：CHEMISTRY

WOS号：WOS:001040391600001

JCR分区：Q1

影响因子：6.7

年份：2023

卷号：95

期号：33

开始页：12487

结束页：12496

文献类型：Article

关键词：　

摘要："Loop-mediatedisothermal amplification (LAMP) is a commonly usedalternative to PCR for point-of-care detection of nucleic acids dueto its rapidity, sensitivity, specificity, and simpler instrumentation.While dual-labeled TaqMan probes are widely used in PCR for single-nucleotidepolymorphism (SNP) genotyping, real-time LAMP primarily relies onturbidimetry or intercalator fluorescence measurements, which canbe non-specific and generate false-positive results. In this study,we propose a closed-tube, dual-labeled RNA-modified probes and RNaseH II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping.Our findings indicate that (1) fluorescence signals were predominantlyderived from probe hydrolysis rather than hybridization, (2) temperature-controlledhybridization between the probe and template ensured the specificityof SNP analysis, and (3) RNase H II hydrolysis between the targetcontaining SNP sites and probes did not exhibit sequence specificity.Our RART-LAMP approach demonstrated excellent performance in genotypingC677T clinical samples, including gDNA extracted from blood, saliva,and swabs. More importantly, saliva and swab samples could be directlyanalyzed without any pretreatment, indicating promising prospectsfor nucleic acid analysis at the point of care in resource-limitedsettings."

基金机构："China Postdoctoral Science Foundation [2021TQ0395]; National Natural Science Foundation of China [82172369, 81972025]"

基金资助正文：We thank all the patients and their families for their kind cooperation. This research was supported by China Postdoctoral Science Foundation (2021TQ0395) and National Natural Science Foundation of China (nos 82172369 and 81972025).