Development of RNA/DNA automated extraction and purification device for infectious disease diagnosis.

Genetic tests using RNA/DNA are the most accurate for diagnosing infectious diseases and assessing disease susceptibility, including COVID-19. However, manual specimen handling and the risk of secondary infections by medical staff highlight the need for automated equipment. Automation methods, such as bead purification, have limitations with high-viscosity specimens, while column purification requires complex equipment. This study aimed to develop an automated device using the column purification method for safe and reliable infectious disease diagnosis. We compared the yield and purification of three nucleic acid extraction methods (centrifugation, pressurization, and depressurization) and examined the adaptation of the extraction methods to automated device. Furthermore, we examined the feasibility of extracting SARS-CoV-2 RNA from COVID-19 patients and using qPCR analysis to determine whether the extraction method could be used as a clinical analyzer. Results varied with different columns and reagents, but pressurization method was selected for the automated device's RNA/DNA extraction. Using an automated device equipped with a pressurization method, RNA extracted from pharyngeal fluids from COVID-19 patients who had already been diagnosed with SARS-CoV-2 by qRT-PCR again tested positive. These findings demonstrate the device's effectiveness for nucleic acid extraction and virus-targeted diagnostics. Moreover, it holds potential for genetic testing in fields like food and environmental measurements. The automated device addresses specimen handling challenges and provides a reliable tool for infectious disease diagnosis.

Detection of rotavirus in clinical specimens using an immunosensor prototype based on the photon burst counting technique.

In this study, a sensitive fluorescence sensor was developed for the detection of small, fluorescence-labeled particles dispersed in a solution. The prototype system comprises of a laser confocal optical system and a mechanical sample stage to detect photon bursting of fluorescence-labeled small particles in sample volumes less than 5 µL within 3 minutes. To examine the feasibility of the prototype system as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. The advantages and disadvantages of this immunosensor with respect to ELISA and RT-PCR, the current gold standards for virus detection, are discussed.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

A pressure-driven column-based technique for the efficient extraction of DNA from respiratory samples.

Currently molecular techniques are a broadly accepted tool for diagnosis and are able to benefit patients in clinical practice. The polymerase chain reaction (PCR) has been especially incorporated into practical applications that are already in widespread use across the globe. With regard to the initial DNA extraction from clinically relevant samples, a number of commercially available kits are commonly used and are also designed to be easy to handle and less labor-intensive. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method using sputum from patients who were diagnosed with pneumonia. Also, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge method, and the compact and automatic nature of the pressure system device provided rapid and valuable information for clinical practice.

Establishment of a reborn MMV-microarray technology: realization of microbiome analysis and other hitherto inaccessible technologies.

BACKGROUND: With the accelerating development of bioscience, the problem of research cost has become important. We previously devised and developed a novel concept microarray with manageable volumes (MMV) using a soft gel. It demonstrated the great potential of the MMV technology with the examples of 1024-parallel-cell culture and PCR experiments. However, its full potential failed to be expressed, owing to the nature of the material used for the MMV chip. RESULTS: In the present study, by developing plastic-based MMVs and associated technologies, we introduced novel technologies such as C2D2P (in which the cells in each well are converted from DNA to protein in 1024-parallel), NGS-non-dependent microbiome analysis, and other powerful applications. CONCLUSIONS: The reborn MMV-microarray technology has proven to be highly efficient and cost-effective (with approximately 100-fold cost reduction) and enables us to realize hitherto unattainable technologies.

Design an easy-to-use infection screening system for non-contact monitoring of vital-signs to prevent the spread of pandemic diseases.

The outbreak of infectious diseases such as influenza, dengue fever, and severe acute respiratory syndrome (SARS) are threatening the global health. Especially, developing countries in the South-East Asia region have been at serious risk. Rapid and highly reliable screening of infection is urgently needed during the epidemic season at mass gathering places, such as airport quarantine facilities, public health centers, and hospital outpatients units, etc. To meet this need, our research group is currently developing a multiple vital-signs based infection screening system that can perform human medical inspections within 15 seconds. This system remotely monitors facial temperature, heart and respiration rates using a thermopile array and a 24-GHz microwave radar, respectively. In this work, we redesigned our previous system to make a higher performance with a user-friendly interface. Moreover, the system newly included a multivariable logistic regression model (MLRM) to determine the possibility of infection. We tested the system on 34 seasonal influenza patients and 35 normal control subjects at the Japan Self-Defense Forces Central Hospital. The sensitivity and specificity of the screening system using the MLRM were 85.3% and 88.6%, respectively.

Development of an infection screening system for entry inspection at airport quarantine stations using ear temperature, heart and respiration rates.

After the outbreak of severe acute respiratory syndrome (SARS) in 2003, many international airport quarantine stations conducted fever-based screening to identify infected passengers using infrared thermography for preventing global pandemics. Due to environmental factors affecting measurement of facial skin temperature with thermography, some previous studies revealed the limits of authenticity in detecting infectious symptoms. In order to implement more strict entry screening in the epidemic seasons of emerging infectious diseases, we developed an infection screening system for airport quarantines using multi-parameter vital signs. This system can automatically detect infected individuals within several tens of seconds by a neural-network-based discriminant function using measured vital signs, i.e., heart rate obtained by a reflective photo sensor, respiration rate determined by a 10-GHz non-contact respiration radar, and the ear temperature monitored by a thermography. In this paper, to reduce the environmental effects on thermography measurement, we adopted the ear temperature as a new screening indicator instead of facial skin. We tested the system on 13 influenza patients and 33 normal subjects. The sensitivity of the infection screening system in detecting influenza were 92.3%, which was higher than the sensitivity reported in our previous paper (88.0%) with average facial skin temperature.