The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths worldwide. However, most SARS-CoV-2 detection methods depend on time-consuming sample preparation and large detection instruments. Herein, a method employing nonbleeding pH paper to achieve both RNA extraction and visual isothermal amplification is proposed, enabling rapid, instrument-free SARS-CoV-2 detection. By taking advantage of capillary forces, pH-paper-based RNA extraction can be accomplished within 1 min without need for any equipment. Further, the pH paper can mediate dye-free visual isothermal amplification detection. In less than a 46-min sample-to-answer time, pH-paper-based extraction and visual detection (termed pH-EVD) can consistently detect 1200 genome equivalents per microliter of SARS-CoV-2 in saliva, which is comparable to TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR). Through coupling with a chemically heated incubator called a smart cup, the instrument-free, pH-EVD-based SARS-CoV-2 detection method on 30 nasopharyngeal swab samples and 33 contrived saliva samples is clinically validated. Thus, the pH-EVD method provides simple, rapid, reliable, low-cost, and instrument-free SARS-CoV-2 detection and has the potential to streamline onsite COVID-19 diagnostics.
Abstract The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐ cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full‐size RNA targets, although LbCas12a exhibits weaker trans‐ cleavage activity than AsCas12a on both single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the “ U niversal N uclease for I dentification of V irus E mpowered by R NA‐ Se nsing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double‐stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.
in our authorship and readership.In the past year, we had authors from across the globe (Figure 3), with China and the United States leading with the highest share of all published articles.We are also thrilled to see that Advanced NanoBiomed Research has readers all over the world, as shown in the country distribution of article downloads (Figure 4).One of the most exciting developments in Advanced NanoBiomed Research in the past year was the acceptance in the Emerging Sources Citation Index (ESCI) database of Web of Science, which is a testament to its quality and impact.
Der Nachweis von Nukleinsäuren spielt eine wichtige Rolle in der medizinischen Diagnostik, der Umweltüberwachung und der Lebensmittelsicherheit. In ihrem Forschungsartikel (e202203826) entwickelten Xue Gao, Yi Zhang und Mitarbeiter einen neuen Biosensor für den amplifikationsfreien Nukleinsäurenachweis, indem sie den trans-Spaltungsmechanismus von Cas13a und ultrasensitive Graphen-Feldeffekttransistoren (gFETs) nutzten. Die Abbildung zeigt die Cas13a-vermittelte RNA-trans-Spaltung auf der gFET-Oberfläche für die Sensorsignalübertragung.
A point-of-care microfluidic device takes unprocessed clinical samples, actively lyses viral RNAs, and performs the target amplification-free nucleic acid detection with a limit of detection down to 10 aM with 25 min sample-to-result time.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have recently received notable attention for their applications in nucleic acid detection. Despite many attempts, the majority of current CRISPR-based biosensors in infectious respiratory disease diagnostic applications still require target preamplifications. This study reports a new biosensor for amplification-free nucleic acid detection via harnessing the trans-cleavage mechanism of Cas13a and ultrasensitive graphene field-effect transistors (gFETs). CRISPR Cas13a-gFET achieves the detection of SARS-CoV-2 and respiratory syncytial virus (RSV) genome down to 1 attomolar without target preamplifications. Additionally, we validate the detection performance using clinical SARS-CoV-2 samples, including those with low viral loads (Ct value >30). Overall, these findings establish our CRISPR Cas13a-gFET among the most sensitive amplification-free nucleic acid diagnostic platforms to date.
Abstract CRISPR‐Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR‐Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR‐Cas12a enzyme, a dual‐mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high‐risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high‐risk pooled HPV subtypes. To enable low‐cost point‐of‐care testing, the assay is incorporated into a paper‐based microfluidic chip. Furthermore, the clinical performance of the paper‐based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.
