Application and Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid SARS-CoV-2 Detection in Humans and Animals

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) requires quick and dependable diagnostic solutions. Our study aimed to optimize the rapid detection of SARS-CoV-2 using the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and to compare its performance with the reverse transcription real-time TaqMan quantitative polymerase chain reaction (RT-qPCR) of RNA extraction protocols. Nasopharyngeal swabs (NPS), oropharyngeal swabs (OPS), mixed nasopharyngeal and saliva samples, and sputum samples from individuals with or without COVID-19 symptoms (outpatient n = 180, inpatient n = 50) were collected and stored at -80 °C. These samples were tested using RT-qPCR, RT-LAMP, and RT-PCR for comparison. Additionally, animal samples were tested, including cattle (n = 20), buffaloes (n = 10), sheep (n = 25), goats (n = 30), horses (n = 5), donkeys (n = 5), mules (n = 3), camels (n = 5), dogs (n = 10), cats (n = 5), and rabbits (n = 6). The detection rates of RT-qPCR were 7.3 ± 0.56, 8.0 ± 0.98, and 5.0 ± 0.56 for nasopharyngeal swabs (NPS), oropharyngeal swabs (OPS), and mixed nasopharyngeal and saliva samples mixed with sputum samples from outpatients (n = 180), respectively. For inpatients (n = 50), the means and SD were 43 ± 0.264, 43 ± 0.245, and 26 ± 0.376, respectively. The sensitivity of RT-LAMP was comparable to RT-qPCR, with 88.9% for outpatients and 99.4% for inpatients. Both assays demonstrated 100% specificity in both groups. The findings demonstrated the direct RT-qPCR assay, both with and without RNA extraction, produced positive results