Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

Azeem Mehmood Butt; Shafiqa Siddique; Xiaoping An; Yigang Tong

For this purpose, ready-to-use master mixes and primer mixes were prepared. Multi-gene approach has previously shown to improve the performance of SARS-CoV-2 PCR assays. The same approach was applied here, and RT-LAMP was designed to simultaneously detect ORF1a and N genes. Recently, several studies have reported LAMP assays for COVID-19. Overall, all these studies highlight the strength of LAMP especially in situations where a rapid and reliable testing is required. Moreover, few commercially developed isothermal systems are already in pipeline for the approval by the relevant authorities however, majority of the reported SARS-CoV-2 LAMP assays were evaluated on synthetic constructs and lack validation on actual samples. Theoretically, such assays shouldn't suffer from any drastic differences, however, the true sensitivity: specificity profile of an assay demands validation on clinical cohort. Therefore, one strength of our assay is validation on COVID-19 positive patients' samples. As given in Table 1 , dual-gene LAMP assay was able to detect 43 out of 45 PCR-confirmed SARS-CoV2 positive samples based upon consensus findings of ORF1a and N genes whereas, none of the non-COVID-19 samples were found to be positive indicating 95% sensitivity and 100% specificity. The two PCR-positive samples designated as sample A (ID#1112-4) and B (ID# 390-3) showed conflicting results. As shown in Figure 1 , sample A showed positive amplification in N assay whereas no amplification was noted with ORF1a assay. On the other hand, sample B showed positive amplification with ORF1a whereas negative according to the N assay. As we considered same output from both assays to assign samples as positive or negative, we concluded these samples as negative. The Cq value of sample B was 36.90 that is indicate of low viral load whereas, sample A had Cq value 31.30. As there was no additional viral RNA available for testing of sample A and B, the negative result of sample A for ORF1a is currently unclear. On the other hand, positive detection of low viral load sample B is indicative that LAMP ORF1a assay is more sensitive than N assay. Evaluation with additional samples is currently underway.