A Rebuilding‐Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N‐in‐1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens

Abstract

Sensitive, rapid and low-cost nucleic acid detection is critical for controlling infectious pathogens.Here, we develop a ready-to-use and multimodal detection based on a rebuilding-free, ultrasensitive and selective strategy named dual hairpin ligation-induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS-CoV, SARS-CoV-2 as model targets, we demonstrate DHLApro provides � zM level ultra-sensitivity, being equaling to 0.45 copy/μL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the "N pathogens-in-1" OR gate screening and accurate multichannel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross-interference between the multiple primer sets is also avoided.

Specific recognition and sensitive quantification of mRNA splice variants via one-pot ligation-dependent loop-mediated isothermal amplification

“…37–39 SYBR Green I, a dsDNA-specific fluorescent intercalator, was employed to perform the real-time measurement in a label-free and cost-effective manner. More importantly, different from our previously developed ligation-dependent RNA sensing strategies, 25–32 the ligation reaction and LAMP are sequentially carried out in one tube, which greatly simplifies the experimental process for mRNA alternative splicing variant analysis and meets the routine measurements of ordinary laboratories.…”

“…4,25 Taking the high specificity of ligases and the exponential nucleic acid replication features of different amplification technologies, many pioneering ligation-mediated methods have been developed for precise analysis of mRNA alternative splicing processes and accurate quantification of splicing variants as well as other RNA targets. 25–32 These ligation-based strategies not only simplify the primer/probe design but also significantly improve the specificity of these methods. However, series open-tube experimental procedures may cause disastrous cross-contamination, leading to false-positive signals, greatly challenging the reliability and accuracy of quantification results.…”

“…Third, the two stem-loop DNA probes for different splicing isoforms are identical except for the anti-target sequences and different target-dependent LAMP can be performed with the universal primers, which can simplify the probe/primer design and reduce the amplification bias. More importantly, compared to the previously developed ligation-based sensing strategies, 25–32 the ligation reaction and LAMP are sequentially carried out and monitored in one tube, which can greatly simplify the experimental process and effectively avoid cross-contamination and false-positive results arising from multiple open-tube operations and testing, 33 which can greatly promote the reliability and accuracy of the proposed one-pot ligation-LAMP.…”

Automated Microfluidic Nucleic Acid Detection Platform-Integrated RPA-T7-Cas13a for Pathogen Diagnosis

“…Isothermal amplification strategies that enable a rapid, inexpensive, and accurate assay offer a promising alternative to PCR for routine molecular testing. , The most common methods include loop-mediated isothermal amplification (LAMP) − and recombinase polymerase amplification (RPA). , Compared to LAMP, RPA requires a more moderate temperature (37–42 °C), and the reaction is faster (10–15 min). Although inexpensive, rapid, and on-site deployable, both techniques suffer from problems associated with nonspecific amplification, resulting in a high rate of false positive diagnoses .…”

Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches

“…Multiplexed detection of nucleic acids is vital for clinical diagnostics and genotyping, and has been made more relevant as a result of the recent COVID-19 pandemic . The analysis of genetic material can provide valuable information for identifying the pathogen type and providing targeted treatment. − Due to the high similarity between the different variants of the same pathogenic species, high specificity is required for bacterial subtyping techniques . Besides the widely used polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and DNA microarray assays, numerous novel nucleic acid detection methods have been developed using nanomaterials and enzymes.…”