Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

ERRATUM: Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Volume 74, no.3, p.214-219, 2021. Page 214, affiliation "1TBA Co., LTD, Sendai; 2Hokkaido University Research Center for Zoonosis Control, Sapporo; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia." should read "1TBA Co., LTD, Sendai, Japan; 2Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; 3Hokkaido University, GI-CoRE Global Station for Zoonosis Control, Sapporo, Japan; 4Zambia National Public Health Institute, Ministry of Health, Lusaka, Zambia; 5Department of Pathology and Microbiology, University Teaching Hospital Ministry of Health, Lusaka, Zambia; and 6Ministry of Health, Ndeke House, Lusaka, Zambia".

Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Despite the availability of anti-tuberculosis drugs, the treatment of tuberculosis has been complicated by drug-resistant tuberculosis. The early detection of drug resistance makes early treatment possible. However, the available tools are mainly for rifampicin resistance detection, and the existing isoniazid resistance detection method is expensive, highly technical, and complicated, making it unsustainable for use in developing nations. This study aimed to develop a simple, rapid, and low-cost diagnostic kit for isoniazid-resistant tuberculosis using the single-stranded tag hybridization method to target an isoniazid resistance-conferring mutation. Specificity and sensitivity were assessed using DNA extracted from 49 isoniazid-resistant and 41 isoniazid-susceptible Mycobacterium tuberculosis clinical isolates cultured in mycobacterial growth indicator tubes. Positive signals were observed on mutant and wild-type lines with 100% sensitivity and specificity compared with Sanger sequencing results. In contrast, no positive signal was observed for non-tuberculosis mycobacteria. The detection limit of this method was 103 CFU or less. The STH-PAS system for isoniazid-resistant M. tuberculosis detection developed in this study offers a better alternative to conventional phenotypic isoniazid resistance determination, which will be of both clinical and epidemiological significance in resource-limited nations.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography.

Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously. LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers. The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.

Evaluation of a rapid diagnostic test for detection of dengue infection using a single-tag hybridization chromatographic-printed array strip format.

A dipstick DNA chromatography assay, a single-tag hybridization-printed array strip (STH-PAS), was evaluated for its efficacy to detect dengue virus (DENV). Reverse-transcribed DNA was amplified by PCR, and the amplified DNA was detected using the STH-PAS system. The method was evaluated using stored RNA samples previously identified to carry all 4 serotypes of dengue, chikungunya, and influenza viruses. Clinical performance was also assessed in a prospective study using plasma from 269 febrile cases from the Emergency Department of St. Luke's Medical Center, Quezon City, Philippines, and 30 afebrile normal healthy volunteers. A Taqman real-time PCR (RT-PCR) assay and a rapid Dengue NS1 test, SD Bioline, were used for comparison. The STH-PAS system was more sensitive in detecting dengue infection compared to Taqman RT-PCR. For DENV serotypes 1, 2, and 3, the detection was 1 to 2 dilutions (10-fold) higher, and for DENV serotype 4, the detection was 2-4 dilutions higher. In clinical studies, the STH-PAS system showed 100% sensitivity with 88.9% and 86.6% specificities compared to Taqman RT-PCR and SD Dengue Duo NS1 test, respectively. The STH-PAS system was found to have a superior sensitivity than the Taqman system. Further evaluation of its performance in the field may provide important data to extend its usefulness for surveillance and epidemiological research in outbreak situations.

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens.

A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.

PCR-dipstick DNA chromatography for profiling of a subgroup of caries-associated bacterial species in plaque from healthy coronal surfaces and periodontal pockets.

The onset of plaque-mediated disease, including dental caries and periodontal diseases, is highly associated with compositional change of the resident microflora from the ecological perspective. As specific bacterial profiles have been linked to different disease stages, microbial compositional measurements might therefore have great value for clinical diagnosis. Previously we have reported a dry-reagent strip biosensor-PCR-dipstick DNA chromatography, which utilized molecular recognition of oligonucleotides and biotin-streptavidin, and the optical property of colored microspheres, for semiquantifying a five-membered subgroup of caries-associated bacterial species in supragingival plaque from healthy coronal surfaces of teeth. The present study aimed to evaluate this technique's ability to differentiate microflora by comparing the subset profiles. Sixteen subgingival plaque specimens were pooled from periodontal pockets and analyzed for the composition of Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces sp. and Veillonella parvula. Detection frequencies, relative abundance of each bacterial species, and the five-membered bacterial profiles were compared between supra- and subgingival groups. The supragingival plaque harbored significantly more of the tested species and higher amount of Actinomyces sp. and V. parvula. In subgingival plaque, the predominance was obscured, since several highly overlapped profiles were found at comparable frequencies. Thus, PCR-dipstick DNA chromatography using the same plaque sample enabled simultaneous profiling of multiple species at species level and facilitated discrimination between anticipated different microflora, making this technique a promising chair-side microbiota profiling method.

A set of external reference controls/probes that enable quality assurance between different microarray platforms.

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration-response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT-PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT-PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.

Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota.

A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria, Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces species, and Veillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.

Tag/hybridization-based sensitive detection of polymerase chain reaction products.

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

A rapid and enhanced DNA detection method for crop cultivar discrimination.

In many crops species, the development of a rapid and precise cultivar discrimination system has been required for plant breeding and patent protection of plant cultivars and agricultural products. Here, we successfully evaluated strawberry cultivars via a novel method, namely, the single tag hybridization (STH) chromatographic printed array strip (PAS) using the PCR products of eight genomic regions. In a previous study, we showed that genotyping of eight genomic regions derived from FaRE1 retrotransposon insertion site enabled to discriminate 32 strawberry cultivars precisely, however, this method required agarose/acrylamide gel electrophoresis, thus has the difficulty for practical application. In contrast, novel DNA detection method in this study has some great advantages over standard DNA detection methods, including agarose/acrylamide gel electrophoresis, because it produces signals for DNA detection with dramatically higher sensitivity in a shorter time without any preparation or staining of a gel. Moreover, this method enables the visualization of multiplex signals simultaneously in a single reaction using several independent amplification products. We expect that this novel method will become a rapid and convenient cultivar screening assay for practical purposes, and will be widely applied to various situations, including laboratory research, and on-site inspection of plant cultivars and agricultural products.

Combinational risk factors of metabolic syndrome identified by fuzzy neural network analysis of health-check data.

BACKGROUND: Lifestyle-related diseases represented by metabolic syndrome develop as results of complex interaction. By using health check-up data from two large studies collected during a long-term follow-up, we searched for risk factors associated with the development of metabolic syndrome. METHODS: In our original study, we selected 77 case subjects who developed metabolic syndrome during the follow-up and 152 healthy control subjects who were free of lifestyle-related risk components from among 1803 Japanese male employees. In a replication study, we selected 2196 case subjects and 2196 healthy control subjects from among 31343 other Japanese male employees. By means of a bioinformatics approach using a fuzzy neural network (FNN), we searched any significant combinations that are associated with MetS. To ensure that the risk combination selected by FNN analysis was statistically reliable, we performed logistic regression analysis including adjustment. RESULTS: We selected a combination of an elevated level of γ-glutamyltranspeptidase (γ-GTP) and an elevated white blood cell (WBC) count as the most significant combination of risk factors for the development of metabolic syndrome. The FNN also identified the same tendency in a replication study. The clinical characteristics of γ-GTP level and WBC count were statistically significant even after adjustment, confirming that the results obtained from the fuzzy neural network are reasonable. Correlation ratio showed that an elevated level of γ-GTP is associated with habitual drinking of alcohol and a high WBC count is associated with habitual smoking. CONCLUSIONS: This result obtained by fuzzy neural network analysis of health check-up data from large long-term studies can be useful in providing a personalized novel diagnostic and therapeutic method involving the γ-GTP level and the WBC count.

Peptide array-based analysis of the specific IgE and IgG4 in cow's milk allergens and its use in allergy evaluation.

Cow's milk (CM) is one of the major causes of food allergies in children. We constructed a peptide array consisting of a linear 16-mer peptide library with an offset of 3-mer, which corresponds to the primary sequences of six major CM allergens. The immune reactivity to cow's milk proteins diminishes with age and clinical tolerance commonly occurs. Although the central role of IgE in allergy is well established, the role of other specific antibody classes in obtaining immunotolerance is not well known. The hypothesis that patients become tolerant when they develop immunological changes particularly with the IgG4 isotype has been proposed. In this study, the binding pattern of the CM protein-specific IgE and IgG4 epitopes was measured using the peptide array with sera of 12 patients with persistent CM allergy (CMA), sera of 5 children who outgrew CMA, and sera of 7 CM-sensitized children without allergy symptoms. In CMA patients the IgG4/IgE fluorescence intensity ratios varied greatly from peptide to peptide, and the scatter plots of IgE versus IgG4 signals using significant IgE-binding peptides showed different distribution patterns. When setting the boundary line based on the IgG4/IgE ratio (IgG4/IgE=2), patients with persistent CMA and CM-sensitized children can be distinguished by the plot pattern of peptides. Furthermore, the number of peptide plots in these regions was less in children who outgrew CMA. The approach employed in this study will allow for the distinction between CMA and CM-sensitization, and will enable the estimation of CMA outgrow by monitoring the time elapsed data.

The use of existing denture-satisfaction ratings for a diagnostic test to indicate prognosis with newly delivered complete dentures.

PURPOSE: The study investigated the relation between subjective satisfaction ratings of existing dentures and outcomes of newly delivered dentures, and the ability of the diagnostic test, using existing ratings, to indicate prognosis with newly delivered dentures. METHODS: Consecutive 165 edentulous patients were recruited from November 2001 to August 2006 at a university-affiliated hospital. Dentures were fabricated with an acrylic base with full-balanced occlusion using hard resin artificial teeth by multiple prosthodontists. At the baseline and 3-month after delivery, patients rate their overall, maxillary, and mandibular satisfaction for existing and replaced dentures on a 100-mm visual analogue scale (VAS). The association between baseline ratings and newly delivered dentures was analyzed by regression analysis. The test's performance was measured by constructing a two-by-two table; patients with the following cutoff values on the VAS (overall:

Development of peptide arrays for detection of IgE-binding epitopes in cow's milk allergens.

Peptide arrays have become versatile tools for high throughput screening assays in biomedical and pharmaceutical research. In this study, we constructed a peptide array that contained linear peptide fragments reported as IgE-binding epitopes for cow's milk allergy (CMA). Various peptides with different solubility in aqueous solutions were dissolved in the buffer solutions containing sodium dodecyl sulfate, and we achieved a consistent spotting of peptide solutions using a piezoelectric ceramic micropump. The IgE-binding patterns were successfully detected by observing the binding of Alexa 647-labeled anti-human IgE using sera from CMA patients. Our technique in this study will provide a potent capability for the development of a peptide array for mapping IgE-epitopes in milk proteins, and it will help researchers better understand the IgE-epitopes associated with the clinical outcome of CMA.