High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor.

False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a "big subtraction" signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/µL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.

Water-soluble polythiophene-based colorimetry for the quick and accurate detection of SARS-CoV-2 RNA.

The SARS-CoV-2-related Corona Virus Disease 2019 (COVID-19) epidemic has had a significant negative impact on society and endangered global health. To quickly stop and constrain the pandemic, a SARS-CoV-2 detection technology that is sensitive, quick and reasonably priced is urgently required. The widely used reverse-transcription polymerase chain reaction (RT-PCR) requires complex equipment and a fair amount of time. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) exhibits significant advantage for early detection of COVID-19 without the requirement for expensive equipment by amplifying a little amount of RNA to a detectable level at isothermal condition. Here, a water-soluble polythiophene-based colorimetric method by combining with RT-LAMP is established for fast and sensitive detection of SARS-CoV-2 RNA. The proposed assay has benefits for the quick detection of SARS-CoV-2 RNA at concentrations as low as 10 aM, or 6 copies/µL.

High-intensity vector signals for detecting SARS-CoV-2 RNA using CRISPR/Cas13a couple with stabilized graphene field-effect transistor

False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a “big subtraction” signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/μL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.

Novel reverse transcription-multiple inner primer loop-mediated isothermal amplification (RT-MIPLAMP) for visual and sensitive detection of SARS-CoV-2.

Since the end of 2019, outbreaks of COVID-19 pandemics have continued in different areas worldwide, which exacerbates the need for rapid, sensitive and simple methods for diagnosis. Currently, COVID-19 diagnosis mainly relies on reverse transcription-polymerase chain reaction (RT-PCR), which requires sophisticated instruments. Reverse transcription-loop mediated isothermal amplification (RT-LAMP), due to its isothermal nature and high specificity, can be used as an alternative. In this paper, a novel visual reverse transcription-multiple inner primer loop-mediated isothermal amplification (RT-MIPLAMP) method is established based on RT-LAMP by adding a pair of inner primers. The RT-MIPLAMP method has a higher sensitivity and shorter reaction time compared with conventional RT-LAMP. By using RT-MIPLAMP, as low as 6 × 103 copies per mL in vitro transcribed (IVT) N gene can be detected within 55 min. Meanwhile, as low as 6 × 104 copies per mL IVT N gene is detectable with conventional RT-LAMP within 80 min. The feasibility of visual RT-MIPLAMP is also validated by detecting the N gene spiked into one healthy volunteer's saliva and the full-length RNA in pseudoviruses, indicating the great potential of visual RT-MIPLAMP for SARS-CoV-2 identification.

One-by-one single-molecule counting method for digital quantification of SARS-CoV-2 RNA.

Digital counting individual nucleic acid molecule is of great significance for fundamental biological research and accurate diagnosis of genetic diseases, which is hard to achieve with existing single-molecule detection technologies. Herein, we report a novel one-by-one single-molecule counting method for digital quantification of SARS-Cov-2 RNA. This method uses one fluorescent micromotor functionalized with peptide nucleic acids (PNAs) to specially capture one target RNA molecule. The RNA-micromotors can be propelled by the electric field to target district and accurately counted. Moreover, the method can also clearly discriminate one-base mutation in the target RNAs, indicating the great potential for clinical diagnostics and virus traceability survey.