Rapid colorimetric detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform: iLACO

Abstract

The recent outbreak of a novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) threatens global health, given serious cause for concern. SARS-CoV-2 is a human-to-human pathogen that caused fever, severe respiratory disease and pneumonia (known as COVID-19). By press time, more than 70,000 infected people had been confirmed worldwide. SARS-CoV-2 is very similar to the severe acute respiratory syndrome (SARS) coronavirus broke out 17 years ago. However, it has increased transmissibility as compared with the SARS-CoV, e.g. very often infected individuals without any symptoms could still transfer the virus to others. It is thus urgent to develop a rapid, accurate and onsite diagnosis methods in order to effectively identify these early infects, treat them on time and control the disease spreading. Here we developed an isothermal LAMP based method-iLACO (isothermal LAMP based method for COVID-19) to amplify a fragment of the ORF1ab gene using 6 primers. We assured the species-specificity of iLACO by comparing the sequences of 11 related viruses by BLAST (including 7 similar coronaviruses, 2 influenza viruses and 2 normal coronaviruses). The sensitivity is comparable to Taqman based qPCR detection method, detecting synthesized RNA equivalent to 10 copies of 2019-nCoV virus. Reaction time varied from 15-40 minutes, depending on the loading of virus in the collected samples. The accuracy, simplicity and versatility of the new developed method suggests that iLACO assays can be conveniently applied with for 2019-nCoV threat control, even in those cases where specialized molecular biology equipment is not available.

Origin, Potential Therapeutic Targets and Treatment for Coronavirus Disease (COVID-19)

“…Recently, some parallel procedures have also been reported for the diagnosis of COVID-19. One of these procedures is loop-mediated isothermal amplification (LAMP), which is a faster single-step procedure, having > 95% sensitivity [52,53]. Further modifications of LAMP-based procedures have been reported, which can reduce the testing time with minimum equipment requirements [54,55].…”

“…The interventions into the ACE2 binding abilities of SARS-CoV-2 and similar viruses, by the inhibition of the corresponding serine protease [106], can be an area of investigation. Easy, highly reliable, and early-stage diagnosis procedures are still required as a challenge for biomedical researchers [52,53]. The presence of SARS-CoV-2 in stool samples of infected individuals raises the question about the fecal-oral transmission of the disease [10].…”

SARS-CoV-2 On-the-Spot Virus Detection Directly from Patients

“…Due to the need for reverse transcription, this method is called reverse-transcribed (RT)-LAMP. Altogether, these advantages eliminate the need for sophisticated lab equipment [5][6][7] . Here, we adjusted RT-LAMP for the detection of SARS-CoV-2 RNA directly from clinical diagnostic swabs of human patients, without RNA purification steps.…”

Detecting SARS-CoV-2 at point of care: Preliminary data comparing Loop-mediated isothermal amplification (LAMP) to PCR

“…Within the past 4-6 weeks, five independent groups have published preprints of submitted manuscripts evaluating novel RT-LAMP testing methods against RT-PCR as gold standard ( Table 4). Two of these used only proven PCR-positive throat and nasal swabs and demonstrated sensitivity >97% for RT-LAMP methods targeting the ORF1ab gene when compared with gold standard RT-PCR [11,12]. Only the study by Yang et al included swabs from both SARS-CoV-2 positive and negative patients and was thus able to produce both a sensitivity and a specificity.…”