Perspectives and Prospects on the application of DNA Aptamer in SARS-CoV-2.

With the SARS-COV-2 spreading worldwide, COVID-19 has induced enormous disaster and challenges to the world public health since 2019. To development effective diagnostic and treatment methods for the SARS-CoV-2 is crucial for the control of the COVID-19 spreading. DNA aptamers, short single DNA oligonucleotides, have shown an immense application potential as molecular probes for early diagnosis and therapy of various diseases. Compared with antibodies, DNA aptamers are easier to synthesize, with high stability and low immunogenicity, which allows it to be widely and rapidly used in various bio-sensing and therapy to against COVID-19 since then. Thus, we here reviewed the development and prospected potential applications of DNA aptamers in diagnosis and treatment of SARS-COV-2.

Reducing COVID-19 diagnostic errors with dNTPαSe supplementation.

Timely and accurate diagnosis of COVID-19 is critical for controlling the pandemic. As the standard method to diagnose SARS-CoV-2, the real-time reverse transcription polymerase chain reaction (RT-qPCR) has good convenience. However, RT-qPCR still has a relatively high false-negative rate, particularly in the case of detecting low viral loads. In this study, using selenium-modified nucleoside triphosphates (dNTPαSe) in the RT-PCR reactions, we successfully increased the detection sensitivity and reduced the false-negative rate in COVID-19 diagnosis. By detecting positive controls, pseudovirus, and clinical samples with the commercial kits, we found that the dNTPαSe supplementation to these kits could generally offer smaller Ct values, permit the viral detection even in single-digit copies, and increase the detection specificity, sensitivity, and accuracy, thereby reducing the false-negative rate. Our experimental results demonstrated that dNTPαSe supplementation can make the commercial kits more specific, sensitive, and accurate, and this method is a convenient and efficient strategy for the disease detection and diagnosis.

Demographic, Virological Characteristics and Prognosis of Asymptomatic COVID-19 Patients in South China.

BACKGROUND: Asymptomatic transmission is a major concern for SARS-CoV-2 community spread; however, little information is available on demographic, virological characteristics and prognosis of asymptomatic cases. METHODS: All COVID-19 patients hospitalized in Guangdong Province from September 1, 2020 to February 28, 2021, were included and were divided into asymptomatic and symptomaticgroup. The source country of all patients, clinical laboratory test results, the genotype of virus and the time of SARS-CoV-2 RNA turning negative or hospitalization were confirmed. RESULTS: Total 233 patients from 57 different countries or regions were included, with 83 (35.6%) asymptomatic and 150 (64.4%) symptomatic patients. Asymptomatic cases were younger (P = 0.019), lower rate in comorbidities (P = 0.021) such as hypertension (P = 0.083) and chronic liver disease (P = 0.045), lower PCT (P = 0.021), DDI (P < 0.001) and ALT (P = 0.029), but higher WBC count (P = 0.002) and lymphocyte (P = 0.011) than symptomatic patients. As for SARS-CoV-2 subtypes, patients infected with B.1.1 (53.8%), B.1.351 (81.8%) and B.1.524 (60%) are mainly asymptomatic, while infected with B, B.1, B.1.1.63, B.1.1.7, B.1.36, B.1.36.1, B.1.36.16, B.1.5 and B.6 were inclined to be symptomatic. Patients infected with variant B.1.351 and B.1.524 spent longer time in SARS-CoV-2 RNA turn negative (26 days, P = 0.085; 41 days, P = 0.007) and hospitalization (28 days, P = 0.085; 43 days, P = 0.004). CONCLUSIONS: The asymptomatic cases are prone to develop in patients with younger age, less comorbidities andinfected with B.1.1 and B.1.524 variants. More attention should be paid for lineage B.1.524 because it can significantly prolong the SARS-CoV-2 RNA negative conversion time and hospitalization in infected cases.

A novel microfluidic RNA chip for direct, single-nucleotide specific, rapid and partially-degraded RNA detection.

Direct RNA detection is critical for providing the RNA insights into gene expression profiling, noncoding RNAs, RNA-associated diseases and pathogens, without reverse transcription. However, classical RNA analysis usually requires RT-PCR, which can cause bias amplification and quantitation errors. To address this challenge, herein we report a microfluidic RNA chip (the microchip prototype) for direct RNA detection, which is primarily based on RNA extension and labeling with DNA polymerase. This detection strategy is of high specificity (discriminating against single-nucleotide differences), rapidity, accuracy, nuclease resistance, and reusability. Further, we have successfully detected disease-associated RNAs in clinical samples, demonstrating its great potentials in biomedical research and clinical diagnosis.

Specificity Enhancement of Deoxyribonucleic Acid Polymerization for Sensitive Nucleic Acid Detection.

Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.

High-quality RT-PCR with chemically modified RNA controls.

In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.