Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment

Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR–based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.     

H6亚型禽流感病毒RT-LAMP检测方法的建立

建立一种快速、敏感、特异的检测H6亚型禽流感病毒(AIV)逆转录环介导等温扩增(RT-LAMP)检测方法.根据H6亚型AIV血凝素(HA)基因序列的保守区8个位点设计了6条特异性LAMP引物,以H6亚型AIV阳性样品RNA为模板进行一步法扩增,对反应条件和反应体系进行优化.结果表明该方法的特异性良好,对其他呼吸道病原体均无扩增反应.该方法灵敏度高,最低可检测到H6亚型AIV RNA为0.01 pg,该方法无需特殊仪器,只需在水浴锅中进行,是一种适于基层的简便、灵敏、快速的H6亚型AIV检测方法.     

改良逆转录环介导等温扩增检测技术快速实时检测H9亚型禽流感病毒

利用改良逆转录环介导等温扩增(RT-LAMP)技术建立一种快速、灵敏的检测方法用于H9亚型禽流感病毒检测。根据H9亚型禽流感病毒血凝素(HA)基因保守区序列中的8个区段设计6条特异性引物,在恒温条件下进行核酸扩增反应,并以琼脂糖凝胶电泳和目视检查绿色荧光两种方法对扩增结果进行判定。结果表明,RT-LAMP的最小检测限为100 fg,灵敏度比PT-PCR高100倍,且与H5亚型、H7亚型禽流感病毒,新城疫病毒(NDV)无交叉反应。目视检查绿色荧光与常规琼脂糖凝胶电泳的判定结果一致。整个扩增检测过程在35 min内即可完成。利用临床样本对RT-LAMP法进行验证,结果与RT-PCR一致。由实时浊度分析得到的标准曲线,计算出临床样本中的病毒质粒拷贝数均在2×107-2×104之间。因此,本研究建立的RT-LAMP方法快速、灵敏、特异性强,是H9亚型禽流感病毒的一种高效检测方法。