A novel paper-based lysis strip for SARS-CoV-2 RNA detection at low resource settings.

Infectious respiratory diseases such as COVID-19 are serious and global concerns from the past to the present. To isolate the spread of infectious diseases even in the absence of a health system, a simple, inexpensive, reliable, sensitive, and selective molecular diagnosis platform for Point of Care Test (POCT) is required. Especially, the nucleic acid extraction step is difficult to perform out of laboratory. Here, we propose a paper-based lysis (PBL) strip for nucleic acid extraction, especially in low-resource settings (LRS). PBL strips are suitable for isolating RNA from viruses with biological interference and inhibitors. We optimized the buffer compositions and membranes of the strip. A simple preparation method using a PBL strip could obtain an eluent for downstream inspection within 20 min. Overall, 104 copies/swaps were detected for 20 min for amplification in combination with Reverse Transcription Loop-Mediated Amplification (RT-LAMP).

Clinical evaluation of an innovative isothermal amplification detection system for COVID-19 diagnosis.

A pre-integrated system design intended for a point-of-care (POC) and sample-to-result diagnostic platform with nucleic acid amplification has been developed, which is equipment/electricity-free without any permanent instruments or manual sample processing. This semi-integrated system focuses on pandemic situations that are suitable for the Affordable, Sensitive, Specific, User-friendly, Robust and rapid, Equipment-free, and Deliverable to the end-user "ASSURED" concept recommended by the World Health Organization (WHO). Nucleic acid amplification is an essential rate-limiting factor in the performance of integrated systems that involve sample preparation and detection. The ORF1ab (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been targeted by RT-LAMP optimization and evaluation using a commercial hot-pack as a heat source that successfully achieves a femto-scale (<6.8 × 102 copies per rxn) limit of detection (LOD) within 40 min (except for the RNA extraction step). Therefore, the prototype system was assessed using COVID-19-suspected clinical samples (eighty eight) and compared with the results of a commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) assay (Allplex SARS-CoV-2 Assay kit (Seegene, Seoul, Republic of Korea)). These innovative approaches achieved over 95% sensitivity and specificity. In conclusion, the developed system using a hot-pack as a heat source is a promising tool that enables the rapid identification of infectious diseases in the real world.

Rapid and simple detection of influenza virus via isothermal amplification lateral flow assay.

Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients.

A simple and innovative sample preparation method for on-site SARS-CoV-2 molecular diagnostics.

Nucleic acid amplification is a widely used diagnostic tool, although it requires a relatively time-consuming and complicated extraction step. To address this issue outside the laboratory, we investigated a sample preparation system and determined that a silica membrane and silica-coated beads are powerful tools for the extraction from raw samples: nucleic acids are kept in the silica membrane, retained during a single wash step, and released at the elution step. The eluent is appropriate for the quantitative real-time polymerase chain reaction (qPCR) and loop-mediated amplification (LAMP) assay in terms of purity and quantity. We also built an innovative equipment-free nucleic acid extraction squeeze system which requires less than 20 min. The sample with improved purity augments the specificity and sensitivity. This system is simple, user-friendly, low-cost, and equipment-free, thus making nucleic acid extraction more accessible and affordable for researchers and untrained users. Furthermore, when combined with the reverse-transcription quantitative real-time polymerase chain reaction method, the method will accelerate the detection of diseases. The same goes when combined with the LAMP assay, especially in developing countries.