Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing a nicking and extension chain reaction system

Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA promoter-containing double-stranded DNA (T7DNA), and thus the final amplification efficiency is not sufficiently high enough to achieve ultrasensitive detection. We herein ingeniously integrate a nicking and extension chain reaction system into the NASBA to establish an ultrasensitive version of NASBA, termed Nicking and Extension chain reaction System-Based Amplification (NESBA). By employing a NESBA primer set designed to contain an additional nicking site at the 5' end of a NASBA primer set, the T7DNA is exponentially amplified through continuously repeated nicking and extension chain reaction by the combined activities of nicking endonuclease (NE) and reverse transcriptase (RT). As a consequence, a much larger number of RNA amplicons would be produced through the transcription of the amplified T7DNA, greatly enhancing the final fluorescence signal from the molecular beacon (MB) probe binding to the RNA amplicon. Based on this unique design principle, we successfully identified the target respiratory syncytial virus A (RSV A) genomic RNA (gRNA) down to 1 aM under isothermal conditions, which is 100-fold more sensitive than regular NASBA

Ultrasensitive Isothermal Detection of SARS-CoV‑2 Based on Self-Priming Hairpin-Utilized Amplification of the G‑Rich Sequence

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints

A personal glucose meter-utilized strategy for portable and label-free detection of hydrogen peroxide

Rapid and precise detection of hydrogen peroxide (H2O2) holds great significance since it is linked to numerous physiological and inorganic catalytic processes. We herein developed a label-free and washing-free strategy to detect H2O2 by employing a hand-held personal glucose meter (PGM) as a signal readout device. By focusing on the fact that the reduced redox mediator ([Fe(CN)6]4-) itself is responsible for the final PGM signal, we developed a new PGM-based strategy to detect H2O2 by utilizing the target H2O2-mediated oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- in the presence of horseradish peroxidase (HRP) and monitoring the reduced PGM signal in response to the target amount. Based on this straightforward and facile design principle, H2O2 was successfully determined down to 3.63 μM with high specificity against various non-target molecules. We further demonstrated that this strategy could be expanded to identify another model target choline by detecting H2O2 produced through its oxidation promoted by choline oxidase. Moreover, we verified its practical applicability by reliably determining extracellular H2O2 released from the breast cancer cell line, MDA-MB-231. This work could evolve into versatile PGM-based platform technology to identify various non-glucose target molecules by employing their corresponding oxidase enzymes, greatly advancing the portable biosensing technologies

Palindromic hyperbranched rolling circle amplification enabling ultrasensitive microRNA detection

We herein describe a palindromic hyperbranched rolling circle amplification (PH-RCA) reaction and its application for ultrasensitive detection of microRNAs (miRNAs). In this strategy, target miRNAs bind to a dumb-bell probe (DP) and initiate the RCA reactions, concomitantly converting the dumb-bell structure to the circular form, which then allows the annealing of the palindromic primers to promote an additional two RCA reactions. As a consequence of the RCA reactions promoted by both target miRNAs and palindromic primers, multiple long concatenated DNA strands would be produced. Importantly, the palindromic primers can also bind to numerous palindromic domains of the long linear single DNA strands, consequently promoting highly branched simultaneous extension reactions at multiple sites. By detecting the fluorescence signals resulting from the amplified DNA products, we successfully identified target miRNA under isothermal conditions with excellent specificity. The PH-RCA technique developed in this work would greatly advance the conventional RCA reaction and HRCA reaction by significantly enhancing the sensitivity and reducing the reaction time within 30 min

Genetic Diversity of Halyomorpha halys (Hemiptera, Pentatomidae) in Korea and Comparison with COI Sequence Datasets from East Asia, Europe, and North America

The brown marmorated stinkbug, Halyomorpha halys, is an invasive insect pest in North America and Europe that attacks crop species and causes substantial economic damage. To evaluate the genetic diversities and distributions of different H. halys populations in East Asia, North America, and Europe, COI sequences obtained from 79 new specimens from Korea and 10 from the USA were compared with 725 existing COI sequences. In total, 45 haplotypes were detected in populations from 10 countries. Sixteen haplotypes from Korea (H34–H49) and 2 from the USA (H50 and H51) were novel. Korean populations exhibited the 2nd highest diversity among the 10 countries, with only Greece exhibiting higher diversity. Haplotype H22 was prominent in Korea, H1 was prominent in China, Greece, Hungary, Italy, Canada, and USA, and H3 was prominent in France and Switzerland. Of the 18 haplotypes found in Korea, 1 was shared with China (H2) and 1 with Greece (H22). Haplotype diversity patterns showed that Korean populations were genetically distinct from populations in China, Europe, and North America. This suggested that populations in Europe and North America arose through multiple invasions from China and that (with the exception of Greece), Korean populations did not spread to other countries. This study represents a comprehensive analysis of H. halys populations in Korea and places these populations in a global context that includes other native populations in East Asia and invasive populations in Europe and North America