Rapid, sensitive, and specific detection of SARS-CoV-2 in nasopharyngeal swab samples of suspected patients using a novel one-step loop-mediated isothermal amplification (one-step LAMP) technique

Abstract

Background In the absence of effective antiviral drugs or vaccines, early and accurate detection of SARS-CoV-2 infection is essential to the COVID-19 pandemic. This study developed and evaluated a novel rapid One-Step LAMP assay to directly detect the SARS-CoV-2 RNA from nasopharyngeal (NP) swab samples of patients with suspected SARS-CoV-2 infection living in deprived areas in comparison to One-Step Real-time PCR. Methods Two hundred fifty-four NP swab samples from patients suspected of COVID-19 infection living in deprived western areas of Iran were tested by TaqMan One-Step RT-qPCR and fast One-Step LAMP assays. Tenfold serial dilutions of SARS-CoV-2 RNA standard strain where the viral copy number in each dilution was previously determined using the qPCR and various templates were used to investigate the analytical sensitivity and specificity of the One-Step LAMP assay in triplicate. Also, the efficacy and reliability of the method compared to TaqMan One-Step RT-qPCR were evaluated using SARS-CoV-2 positive and negative clinical samples. Results The results of the One-Step RT-qPCR and One-Step LAMP tests were positive in 131 (51.6%) and 127 (50%) participants, respectively. Based on Cohen’s kappa coefficient (κ), the agreement between the two tests was 97%, which was statistically significant (P < 0.001). The detection limit for the One-Step LAMP assay was 1 × 101 copies of standard SARS-CoV-2 RNA per reaction in less than an hour in triplicates. Negative results in all samples with non-SARS-CoV-2 templates represent 100% specificity. Conclusions The results showed that the One-Step LAMP assay is an efficient consistent technique for detecting SARS-CoV-2 among suspected individuals due to its simplicity, speed, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease epidemic control, timely treatment, and public health protection, especially in poor and underdeveloped countries.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2

“…According to the obtained results, it seems that the RT-LAMP method was able to detect SARS-CoV-2 carriers, one week before emerging the clinical symptoms, while the RT-qPCR method is not able to detect carriers. In this study, there was no significant relationship between age and SARS-CoV-2 infection that was consistent with the results of the studies by Khanizadeh et al [ 32 ], and Novosad et al [ 41 ]. While in contrary to this a significant relationship between age and SARS-CoV-2 infection was reported in the study of XU et al [ 42 ].…”

“…Based on Cohen’s kappa test, the agreement rate between LAMP and RT-qPCR tests in the nasopharyngeal sample was 94% (27.4% and 27.1% positivity rate respectively) and 93% in the saliva sample (26.8% and 25.1% positivity rate respectively) which, was statistically significant ( P < 0.001). The results were consistent with the results in the study by Khanizadeh et al [ 32 ] in terms of the agreement between the two techniques showing the accuracy and reliability of the results of the One-Step LAMP technique compared to the One-Step RT-qPCR method. Several studies have reported that the LAMP technique is a simple, fast, and sensitive isothermal amplification technique.…”

“…Moreover, the LAMP method has demonstrated great success in identifying and diagnosing SARS-CoV-2 infection in clinical samples from patients because of highly specific on six independent primers to recognize the target sequence [ 33 , 34 ]. In this study, it was shown that the One-Step LAMP technique was able to detect 1×10 1 copies of the standard RNA of the SARS-CoV-2 virus, which shows the high sensitivity of this technique so the results were consistent with the results in the studies of Khanizadeh et al [ 32 ]. The high sensitivity of this technique can be attributed to the use of two forward and reverse loop primers (LF and LB primers), which act as an accelerator of the replication reaction, reducing the reaction time and increasing the detection speed.…”

Molecular Point‐of‐Care Assay Development: Design and Considerations

“…Several studies evaluated and proved the high sensitivity and specificity of LAMP technology. In Khanizadeh et al (2023), the specificity and sensitivity of the one-step LAMP technique were evaluated for the detection of the SARS-CoV-2 virus from nasopharyngeal swabs without extracting viral RNA. The study demonstrates that the assay can detect 1 × 10 1 copies of SARS-CoV-2 RNA strands, showing the high sensitivity of this technique.…”

“…Optimally, the target DNA region should range between 130 to 300 bp to be amplified by LAMP (Das et al, 2022). LAMP is capable of targeting different, unique regions of the target sequence using multiple primers during the same reaction (Khanizadeh et al, 2023). Furthermore, LAMP is compatible with multiplexing; for example, Zhu et al (2020) developed a multiplex RT-LAMP coupled with a nanoparticle-based lateral flow biosensor (LFB) to diagnose COVID-19, where two genes of SARS-Cov-2 virus the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) are amplified simultaneously in a single-tube reaction.…”