A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point-of-care

Abstract

The COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment.In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-touse kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

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“…100,101 This method (Figure 2E) was designed to directly amplify specific reversely transcripted cDNA through incubation at a fixed temperature (65 C) through only one-step reverse transcription and achieved an efficiency of 10 9 amplicons of genomic DNA in less than 1 h. It has been demonstrated that RT-LAMP can provide a sensitivity 10-fold higher than that of qRT-PCR. 102 Recently, RT-LAMP-based assays have been deployed for rapid and specific detection of pathogenic avian influenza and respiratory viruses (including SARS-CoV-2 [103][104][105] and other infectious diseases in humans, [102][103][104][106][107][108][109] and have also been successfully integrated into several LionX platforms. [110][111][112][113][114] In contrast to RT-LAMP that utilizes only one enzyme such as DNA polymerase for target NA amplification, other commonly used isothermal amplification methods require several enzymes and proteins for the amplification process.…”

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“…Most reports achieved the clinical sensitivity and specificity of RT-LAMP within 75–100% and 98.7–100%, respectively, while the LOD ranged from 1 to 304 copies per reaction [ 127–134 ]. Another advantage that makes LAMP fit for POCT is the use of lyophilized reagents without sacrificing quality [ 135 ], which expands the kit shelf-life to years at 4°C or several weeks at room temperature [ 136 , 137 ]. Nevertheless, LAMP performance is heavily dependent on its custom design and might not yet be comparable to qRT-PCR in some cases, as it was reported to be reliable up to the viral load equivalent of Ct (cycle threshold) < 30 [ 131 ], which was in line with the observations from other groups [ 138 , 139 ].…”

“…Tran et al . found that RT-LAMP is superior to the other two iNAATs that utilize Bst DNA polymerase for detecting SARS-CoV-2, cross-priming amplification (CPA), and polymerase spiral reaction (PSR) with a 20–40 times lower LOD value [ 135 ]. Naveen and colleagues showed that the LOD of RT-LAMP was equal or one order of magnitude lower than that of RT-RPA in detecting two ginger-infecting viruses [ 152 ] and cardamom vein clearing virus [ 153 ].…”

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“…Urine samples showed more significant signals, and no cross-reactivity. When three colorimetric isothermal amplification methods [LAMP, cross-priming amplification (CPA), and polymerase spiral reaction (PSR)] were compared, LAMP showed the best results in naso- and oropharyngeal swabs with the highest sensitivity (43 copies) ( 50 ). LAMP or RT-LAMP based techniques are highly successful in SARS-CoV-2 detection.…”