A novel isothermal method for amplification of long specific amplicon from linear template.

Isothermal nucleic acid amplification methods have been successfully developed and applied for diagnostic purpose, especially for detection of pathogens. However, amplicon size of such methods is relatively short (< 500 bp) to limit their application for long amplicon production that can be used for various downstream applications including genomic surveillance of pathogens. To fill the gap, we developed a method for specific amplification of kilobases-long target sequence from RNA templates. This method, named CREA, utilizes sequence specific recombination of Cre recombinase to generate circular intermediate template for subsequent RCA reaction. CREA with SARS-CoV-2 spike template could amplify ~ 2.9 kb target and up to ~ 1.9 kb amplicon was able to produce in sufficient amount for general cloning. Each step of CREA procedure was thoroughly analyzed to provide directions for further optimizations. Furthermore, we evaluated a variation of CREA which utilized DNA ligase.

Development of Three-Stage Bioaerosol Sampler for Size-Selective Sampling.

In this study, a three-stage bio-aerosol sampler with a sampling flow rate of 170 L/min was designed and fabricated for sampling the bio-aerosols released during human breathing and coughing, and its performance was evaluated. The sampler was constructed using a cyclone separator with a cutoff size of 2.5 µm as a preseparator, a multinozzle virtual impactor with a cutoff size of 0.34 µm as an aerosol concentrator, and a Bio-Sampler, which is a commercial product, for collecting bio-aerosols in a collection fluid. The collection efficiency of the sampler was evaluated through simulations and experiments. Only particles with sizes of 0.1-4 µm were selectively collected in the collection fluid. Bacteriophage bio-aerosols were sampled using the developed sampler and ACD-200 Bobcat sampler, which is a commercial product. The amounts of collected bacteriophages were compared using the polymerase chain reaction (PCR) technique. The sampling performance of the developed sampler was similar to that of the ACD-200 Bobcat sampler. Moreover, the developed sampler showed its ability to sample bio-aerosols of a specific size range and collect them directly in a collection fluid for the PCR analysis. Therefore, the developed sampler is expected to be useful for indoor environmental monitoring by effectively sampling the bio-aerosols released indoors during human breathing and coughing.

High-volume sampler for size-selective sampling of bioaerosols including viruses.

Owing to the recent global spread of the new coronavirus SARS-CoV-2, the development of technology to effectively detect viruses in crowded public places is urgently needed. In this study, a three-stage high-volume bioaerosol sampler was developed for the size-selective sampling of bioaerosols through the suction of air at a high flow rate of 1000 L/min. In stage 1, an omnidirectional inlet cyclone separator that can draw air from all directions was applied to collect bioaerosols larger than 10 µm in the collection fluid. In stage 2, an axial flow cyclone separator was used to collect bioaerosols sized between 2.5 and 10 µm in the collection fluid. In stage 3, bioaerosols smaller than 2.5 µm were collected on a filter and extracted in a solution through an elution process using a sodium phosphate buffer. To simulate the suspension of bioparticles including viruses that are attached to other particles in the atmosphere, the aerosol samples were prepared by coagulating aerosolized bacteriophages with Arizona test dust. Then, the coagulated particles were collected for 30 min using the developed bioaerosol sampler, and the samples collected in each stage were analyzed via polymerase chain reaction (PCR) method. The PCR analysis results confirmed that the high-volume bioaerosol sampler enables size-selective bioaerosol sampling even at a high airflow rate of 1000 L/min. The developed high-volume bioaerosol sampler will be useful in detecting viruses through PCR analysis because it can collect bioaerosols within a specific size range.

Comparison of Digital PCR and Quantitative PCR with Various SARS-CoV-2 Primer-Probe Sets.

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Comparative Analysis of Primer-Probe Sets for RT-qPCR of COVID-19 Causative Virus (SARS-CoV-2).

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within 8 months of the outbreak, more than 10,000,000 cases of COVID-19 have been confirmed worldwide. Since human-to-human transmission occurs easily and the rate of human infection is rapidly increasing, sensitive and early diagnosis is essential to prevent a global outbreak. Recently, the World Health Organization (WHO) announced various primer-probe sets for SARS-CoV-2 developed at different institutions: China Center for Disease Control and Prevention (China CDC, China), Charité (Germany), The University of Hong Kong (HKU, Hong Kong), National Institute of Infectious Diseases in Japan (Japan NIID, Japan), National Institute of Health in Thailand (Thailand NIH, Thailand), and US CDC (USA). In this study, we compared the ability to detect SARS-CoV-2 RNA among seven primer-probe sets for the N gene and three primer-probe sets for the Orf1 gene. The results revealed that "NIID_2019-nCOV_N" from the Japan NIID and "ORF1ab" from China CDC represent a recommendable performance of RT-qPCR analysis for SARS-CoV-2 molecular diagnostics without nonspecific amplification and cross-reactivity for hCoV-229E, hCoV-OC43, and MERS-CoV RNA. Therefore, the appropriate combination of NIID_2019-nCOV_N (Japan NIID) and ORF1ab (China CDC) sets should be selected for sensitive and reliable SARS-CoV-2 molecular diagnostics.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.

Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic materials. Here, we provide a protocol to perform reverse transcription LAMP targeting SARS-CoV-2. We adopted both real-time fluorescence detection and end-point colorimetric detection approaches. Our protocol would be useful for screening diagnosis of COVID-19 and be a baseline for development of improved POCT NAAT.

LATS-YAP/TAZ controls lineage specification by regulating TGFß signaling and Hnf4α expression during liver development.

The Hippo pathway regulates the self-renewal and differentiation of various adult stem cells, but its role in cell fate determination and differentiation during liver development remains unclear. Here we report that the Hippo pathway controls liver cell lineage specification and proliferation separately from Notch signalling, using mice and primary hepatoblasts with liver-specific knockout of Lats1 and Lats2 kinase, the direct upstream regulators of YAP and TAZ. During and after liver development, the activation of YAP/TAZ induced by loss of Lats1/2 forces hepatoblasts or hepatocytes to commit to the biliary epithelial cell (BEC) lineage. It increases BEC and fibroblast proliferation by up-regulating TGFß signalling, but suppresses hepatoblast to hepatocyte differentiation by repressing Hnf4α expression. Notably, oncogenic YAP/TAZ activation in hepatocytes induces massive p53-dependent cell senescence/death. Together, our results reveal that YAP/TAZ activity levels govern liver cell differentiation and proliferation in a context-dependent manner.

An evolutionarily conserved negative feedback mechanism in the Hippo pathway reflects functional difference between LATS1 and LATS2.

The Hippo pathway represses YAP oncoprotein activity through phosphorylation by LATS kinases. Although variety of upstream components has been found to participate in the Hippo pathway, the existence and function of negative feedback has remained uncertain. We found that activated YAP, together with TEAD transcription factors, directly induces transcription of LATS2, but not LATS1, to form a negative feedback loop. We also observed increased mRNA levels of Hippo upstream components upon YAP activation. To reveal the physiological role of this negative feedback regulation, we deleted Lats2 or Lats1 in the liver-specific Sav1-knockout mouse model which develops a YAP-induced tumor. Additional deletion of Lats2 severely enhanced YAP-induced tumorigenic phenotypes in a liver specific Sav1 knock-out mouse model while additional deletion of Lats1 mildly affected the phenotype. Only Sav1 and Lats2 double knock-down cells formed larger colonies in soft agar assay, thereby recapitulating accelerated tumorigenesis seen in vivo. Importantly, this negative feedback is evolutionarily conserved, as Drosophila Yorkie (YAP ortholog) induces transcription of Warts (LATS2 ortholog) with Scalloped (TEAD ortholog). Collectively, we demonstrated the existence and function of an evolutionarily conserved negative feedback mechanism in the Hippo pathway, as well as the functional difference between LATS1 and LATS2 in regulation of YAP.