Gamil S.G. Zeedan^1*,Abeer M. Abdalhamed² ,Amel M. Naguib³, Said A. Shalaby ⁴, Mona A. M. Awad⁵,  Mervat Ibrahim⁶

1; 2: Parasitology and Animals Diseases Department (Virology Unit) National Research Centre, 33 Bohouth Street, Dokki, 12622, Giza, Egypt.

3: Virology Department, Central Public Health Laboratories, Ministry of Health and Population, Cairo Egypt

4: Tropical Medicine, Gastroenterology & Hepatology- Digestive Endoscopy at Complementary Medicine Department, Medical Research division National Research Centre, 33 Bohouth Street, Dokki, 12622, Giza, Egypt.

5: Clinical and Chemical Pathology Department , Medical Research division National Research Centre, 33 Bohouth Street, Dokki, 12622, Giza, Egypt.

6: Virology Department, Animal Health Research Institute (AHRI), Nadi El-Said Street, Dokki, Giza, Egypt.

Abstract

Background:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19) pandemic and put global pressure on modern societies, particularly the infrastructure related to health care. Therefore, diagnostic tests play an important role in containing COVID-19, allowing rapid implementation of control measures that restrict the spread by detecting and isolating cases as well as detecting contact to people with an infected patient, thereby preventing the spread of infection around the world.

Objectives:

Our project was aimed at using nanotechnology applications to provide smart solutions for SARS-CoV-2 virus detection with high sensitivity, specificity, accuracy, effectiveness, reliability, and low cost. I. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a nanoparticle was used to detect triple genes to rapidly diagnose SARS-CoV-2 in a one-step, single-tube reaction. Our project developed Aptamer  lateral flow assay devices (Apta-LFA), which are inexpensive, easy to use, but have to be verified, as they are rather unspecific and can produce both, false positive and false negative results. Our objective was to combine the speed of Apta-LFA for SARS-CoV-2 with the reliability of the RT-qPCR test. Amplification was performed using RT-qPCR and RT-LAMP.

Methodology:

Rapid diagnostic tests were created for the detection of SARS-CoV2 and compared to Q RT-PCR and RT- LAMP assays in random sample specimens with or without RNA extraction kits from nasopharyngeal (NP) and oropharynxes (OP) of people (outpatient n = 180, and inpatient storage samples n = 50) with or without SARS-CoV-2 clinical symptoms by physician staff in the virology laboratory of the Ministry of Health in Egypt. The second diagnostic point was the optimized RT-LAMP assay for detection of SARS-CoV-2 in different sample specimens in heat block and thermocycler machines at 66 C for 30 minutes using the RNA extraction Kit or directly from the sample specimen (on-heated, heated at 95 for 5 min and 10 min). Also, we developed a novel method based on gold nanoparticles conjugated with DNA aptamer to detect SARS-CoV-2 N and spike proteins, which may be a potential early diagnostic tool for SARS-CoV-2 detection. As well as it may be used as a treatment for SARS-CoV-2, it is further considered for use in antiviral therapy by interfering with the N protein function.

Results & findings:

We used the RT-LAMP assay for the detection of SARS-CoV-2 in different sample specimens. It was performed using a heat block and thermocycler machine at 66 °C for 30 min with an RNA extraction kit or direct extraction from the sample specimen (non-heated or heated at 95 °C for 5 and 10 min). The highest detection rate of the SARS-Co2 RNA by RT-qPCR from the different samples, NPS, OPS, mixed NOP, and sputum, was 4.44%, 3.88%, 4.44%, and 8.33%, respectively. While the positive percentage of inpatient samples in NPS and OPS was 80% and 84%, respectively. Nine out of 180 nasopharyngeal swabs from outpatients were confirmed to be positive by RT-qPCR and RT-LAMP, while 42 nasopharyngeal swabs out of 50 nasopharyngeal and or pharyngeal swabs stored at -80 o C were confirmed to be positive samples. The sensitivity of RT-LAMP was 88.9% and 100%. Its specificity was 99.4% and 100 (at P ≤ 0.05 and CI = 95%) in outpatients and inpatients samples, respectively. The sensitivity of the direct method was sufficient to reliably detect patients who carried SARS-CoV-2.

Conclusions:

We can conclude that the direct RT-PCR and RT qPCR without RNA ‎isolation gave similar results to RNA extraction by RNA extraction kits.  Also, Furthermore, RT-LAMP was a rapid, simple, specific, and sensitive assay for SARS-CoV-2 detection from clinical samples and will be a powerful tool for SARS-CoV-2 identification and the monitoring of individuals with a suspicion of SARS-V infection. We have developed a novel method based on ssDNAaptamer for the detection of SARS-CoV-2 N protein, which may have potential for the early diagnosis of SARS-CoV-2. Given that N protein is also required for viral genome assembly and host immune response antagonism, However, extensive research and validation by testing several hundred samples are necessary to recommend this assay to clinical diagnostic laboratories. The current research provided smart solutions for the rapid diagnosis of SARS-CoV-2, which is the causative agent of coronavirus disease 2019 (COVID-19) with high sensitivity, specificity, accuracy, and reliability at a low cost.