Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes.

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

The Long-Term Impact of COVID-19 Pneumonia on the Pulmonary Function of Survivors.

BACKGROUND: The sequelae of COVID-19 pneumonia on pulmonary function and airways inflammation are still an area of active research. OBJECTIVE: This research aimed to explore the long-term impact of COVID-19 pneumonia on the lung function after three months from recovery. METHODS: Fifty subjects (age 18-60 years) were recruited and classified into two groups: the control group (30 subjects) and the post-COVID-19 pneumonia group (20 patients). Pulmonary function tests, spirometry, body plethysmography [lung volumes and airway resistance (Raw)], diffusion capacity for carbon monoxide (DLCO), and fractional exhaled nitric oxide (FeNO), were measured after at least 3 months post-recovery. RESULTS: Significant reduction in total lung capacity (TLC), forced vital capacity (FVC), forced expiratory volume (FEV1), FEV1/FEV, and diffusing capacity for carbon monoxide (DLCO) was observed in post-COVID-19 subjects compared to controls. Restrictive lung impairment was observed in 50% of post-COVID-19 cases (n = 10) compared to 20% in the control group (n = 6, P = 0.026). In addition, mild diffusion defect was detected in 35% (n = 7) of the post-COVID-19 group compared to 23.3% (n = 7) in the controls (P = 0.012). CONCLUSION: COVID-19 pneumonia has an impact on the lung functions in terms of restrictive lung impairment and mild diffusion defect after three months from recovery. Therefore, a long-term follow-up of the lung function in post-COVID-19 survivors is recommended.

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.

Coronavirus diseases 2019: Current biological situation and potential therapeutic perspective.

Coronavirus Disease 2019 (COVID-19) caused by a Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first reported in Wuhan, China at the end of December 2019. SARS-CoV-2 is a highly pathogenic zoonotic virus and closely related to the Severe Acute Respiratory Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The COVID-19 was declared as a global pandemic due to its high infectiousness, and worldwide morbidities and mortalities. The Chinese scientists at the start of the outbreak reported genome sequences, which made the characterization of glycoproteins and other structural proteins possible. Moreover, researchers across the world have widely focused on understanding basic biology, developing vaccines, and therapeutic drugs against the COVID-19. However, until now, no promising treatment options, as well as vaccines, are available. In this review, we have described SARS-CoV-2's genome, transmission, and pathogenicity. We also discussed novel potential therapeutic agents that can help to treat the COVID-19 patients.