Detection of thermotolerant coliforms and SARS-CoV-2 RNA in sewage and recreational waters in the Ecuadorian coast: A call for improving water quality regulation.

Wastewater surveillance represents an alternative approach to regulating contamination and the early detection of infectious agents and outbreaks of diseases of public health importance. This study evaluated domestic wastewater effects on recreational waters in estuarine and seawater bodies in Guayas and Santa Elena provinces in Ecuador, South America. Fecal indicator bacteria (thermotolerant coliforms) served as key indicators for evaluation. Physical, chemical, and microbiological quality markers following the Ecuadorian environmental quality standard and the discharge of effluents to the water resource were analyzed. Samples were collected from 44 coastal sites and 2 oxidation lagoons during the dry and rainy seasons of 2020 and 2021, respectively. SARS-CoV-2 RNA was detected in samples with higher E. coli concentrations using reverse transcription quantitative PCR to detect the genes N and ORF1ab. All samples analyzed for SARS-CoV-2 showed Ct ˂ 40 for at least one gene. Four samples showed at least 20 genome copies of gene N per reaction. These were at an artisanal fishing port, an estuarine area (Palmar), a recreational bay, and an oxidation lagoon. A moderate correlation was found between SARS-CoV-2 RNA, thermotolerant coliform and E. coli (p-value ≤ 0.0037), and a strong and positive correlation between thermotolerant coliform and E. coli. (p-value ≤ 0.00001), highlighting the utility of these established parameters as a proxy of the virus. Significant differences were found in the concentrations of thermotolerant coliforms between seasons (p-value = 0.016) and sites (p-value = 0.005). The highest levels of coliforms were found in the dry season (63000 MPN/100 mL) in Anconcito and during the rainy season (14000 MPN/100 mL) at Esterillo in Playas County. It is recommended that the decentralized autonomous governments of the surveyed provinces in Ecuador implement urgent corrective actions and establish medium-term mechanisms to minimize a potential contamination route. Additional parameters must be included in the monitoring, such as Enterococcus and intestinal parasites, due to their public health implications. In the oxidation lagoons, maintenance actions must be carried out, including the dissolution of sediments, an increase in water retention times, and in situ treatment of the sludge, to improve the system's performance.

An Immiscible-Phase Filtration Device for Isolation, Amplification, and Detection of Nucleic Acids for Clinical Diagnostics.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: An extensive analysis based on sequencing parameters.

During the SARS-CoV-2 pandemic, many countries established wastewater (WW) surveillance to objectively monitor the level of infection within the population. As new variants continue to emerge, it has become clear that WW surveillance is an essential tool for the early detection of variants. The EU Commission published a recommendation suggesting an approach to establish surveillance of SARS-CoV-2 and its variants in WW, besides specifying the methodology for WW concentration and RNA extraction. Therefore, different groups have approached the issue with different strategies, mainly focusing on WW concentration methods, but only a few groups highlighted the importance of prefiltering WW samples and/or purification of RNA samples. Aiming to obtain high-quality sequencing data allowing variants detection, we compared four experimental conditions generated from the treatment of: i) WW samples by WW filtration and ii) the extracted RNA by DNase treatment, purification and concentration of the extracted RNA. To evaluate the best condition, the results were assessed by focusing on several sequencing parameters, as the outcome of SARS-CoV-2 sequencing from WW is crucial for variant detection. Overall, the best sequencing result was obtained by filtering the WW sample. Moreover, the present study provides an overview of some sequencing parameters to consider when optimizing a method for monitoring SARS-CoV-2 variants from WW samples, which can also be applied to any sample preparation methodology.

Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Porous Agarose Layered Magnetic Graphene Oxide Nanocomposite for Virus RNA Monitoring in Wastewater.

The detection of virus RNA in wastewater has been established as a valuable method for monitoring Coronavirus disease 2019. Carbon nanomaterials hold potential application in separating virus RNA owing to their effective adsorption and extraction capabilities. However, carbon nanomaterials have limited separability under homogeneous aqueous conditions. Due to the stabilities in their nanostructure, it is a challenge to efficiently immobilize them onto magnetic beads for separation. Here, we develop a porous agarose layered magnetic graphene oxide (GO) nanocomposite that is prepared by agglutinating ferroferric oxide (Fe3O4) beads and GO with agarose into a cohesive whole. With an average porous size of approximately 500 nm, the porous structure enables the unhindered entry of virus RNA, facilitating its interaction with the surface of GO. Upon the application of a magnetic field, the nucleic acid can be separated from the solution within a few minutes, achieving adsorption efficiency and recovery rate exceeding 90% under optimized conditions. The adsorbed nucleic acid can then be preserved against complex sample matrix for 3 days, and quantitatively released for subsequent quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. The developed method was successfully utilized to analyze wastewater samples obtained from a wastewater treatment plant, detecting as few as 10 copies of RNA molecules per sample. The developed aMGO-RT-qPCR provides an efficient approach for monitoring viruses and will contribute to wastewater-based surveillance of community infections.

Preamplification-free ultra-fast and ultra-sensitive point-of-care testing via LwaCas13a.

CRISPR based nucleic acid detection technology provides a deployable approach to point of care testing. While, there remain challenges limiting its practical applications, such as the need for pre-amplification and the long turnaround time. Here, we present a self-cascade signal amplification method with LwaCas13a and an artificially designed "U" rich RNA of stem-loop structure (URH) for pre-amplification-free ultra-fast and ultra-sensitive point-of-care testing (PASSPORT). The PASSPORT system contains: URH, crRNA targeted the URH, crRNA targeted the interesting RNA, fluorescent RNA reporter and LwaCas13a. The assay realized the detection of 100 copies/mL, within 5 min. The PASSPORT platform was further adopted for the detection of marker gene from SASR-CoV-2 and Severe fever with thrombocytopenia syndrome virus (SFTSV), respectively, and 100% accuracy for the analysis of clinical specimens (100 SASR-CoV-2 specimens and 16 SFTSV specimens) was obtained. Integrated with a lateral flow assay device, this assay could provide an alternative platform for the development of point of care testing (POCT) biosensors. PASSPORT has the potential to enable sensitive, specific, user-friendly, rapid, affordable, equipment-free and point-of-care testing for the purpose of large-scale screening and in case of epidemic outbreak.

Development of a novel Colorimetric Assay for the rapid diagnosis of Coronavirus disease 2019 from nasopharyngeal samples.

Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25 copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15 copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30 min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.

A 17-month longitudinal surface sampling study carried out on public transport vehicles operating in England during the COVID-19 pandemic identified low levels of SARS-CoV-2 RNA contamination.

AIMS: To monitor severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA contamination in vehicles operating in England during the pandemic, to better understand transmission risk of SARS-CoV-2 on public transport. METHODS AND RESULTS: We collected 1314 surface samples between December 2020 and April 2022 on trains and buses managed by five different transport operators. The presence of SARS-CoV-2 RNA was investigated through reverse transcription polymerase chain reaction (RT-PCR). SARS-CoV-2 RNA was found on 197 (15%) of the 1314 surfaces sampled, including seat head rests, handholds, and air extract grilles, but the levels of RNA recovered on those samples (median value of 23.4, interquartile range: 14.3-35.4, N gene copies per extraction) made the presence of infectious virus at the time of sampling extremely unlikely. However, detection rates varied over time with peaks broadly coinciding with times of high community transmission, when it was more likely that people infected with SARS-CoV-2 were travelling on public transport. CONCLUSION: During the pandemic, and as in other public spaces, low levels of SARS-CoV-2 RNA were found on surfaces associated with public transport.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

Development of a self-contained microfluidic chip and an internet-of-things-based point-of-care device for automated identification of respiratory viruses.

The COVID-19 pandemic greatly impacted the in vitro diagnostic market, leading to the development of new technologies such as point-of-care testing (POCT), multiplex testing, and digital health platforms. In this study, we present a self-contained microfluidic chip integrated with an internet-of-things (IoT)-based point-of-care (POC) device for rapid and sensitive diagnosis of respiratory viruses. Our platform enables sample-to-answer diagnostics within 70 min by automating RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescence detection. The microfluidic chip is designed to store all the necessary reagents for the entire diagnostic assay, including a lysis buffer, a washing buffer, an elution buffer, and a lyophilized RT-LAMP cocktail. It can perform nucleic acid extraction, aliquoting, and gene amplification in multiple reaction chambers without cross-contamination. The IoT-based POC device consists of a Raspberry Pi 4 for device control and data processing, a CMOS sensor for measuring fluorescence signals, a resistive heater panel for temperature control, and solenoid valves for controlling the movement of on-chip reagent solutions. The proposed device is portable and features a touchscreen for user control and result display. We evaluated the performance of the platform using 11 clinical respiratory virus samples, including 5 SARS-CoV-2 samples, 2 influenza A samples, and 4 influenza B samples. All tested clinical samples were accurately identified with high specificity and fidelity, demonstrating the ability to simultaneously detect multiple respiratory viruses. The combination of the integrated microfluidic chip with the POC device offers a simple, cost-effective, and scalable solution for rapid molecular diagnosis of respiratory viruses in resource-limited settings.

Evaluation of the SARS-CoV-2 RNA detection reagent LAMPdirect Genelyzer KIT using nasopharyngeal swab and saliva samples.

The LAMPdirect Genelyzer KIT allows for the detection of SARS-CoV-2 RNA in saliva samples with a loop-mediated isothermal amplification (LAMP) method and generates results within 20 min. It has been approved by the Pharmaceuticals and Medical Devices Agency in Japan. In this study, the performance of the LAMPdirect Genelyzer KIT was compared with that of the RT-qPCR reference method using 50 nasopharyngeal swabs and 100 saliva samples. In addition, we evaluated the applicability of an alternative reverse transcriptase and the effects of an inactivation buffer. The total agreement rates were 80.0 % and 82.0 % for nasopharyngeal and saliva samples, respectively. When considering samples at the detection limit (50 copies/reaction) that increases the chance of transmission between humans, the total agreement rates were 100% and 94.1% for nasopharyngeal and saliva samples, respectively. The LAMP method is simple, fast, and inexpensive, making it useful for small medical institutions or rural areas.

Evaluation of an automated platform for the detection of HEV RNA in plasma and stool.

INTRODUCTION: We evaluated the performance of the automated Altostar HEV RNA platform for detecting HEV RNA. METHODS AND RESULTS: Clinical performance was determined by testing 81 plasma samples and 10 fecal samples manually quantified previously with the Realstar RT-PCR assay using the Magnapure instrument for extraction. The assays were concordant for 79/81 plasma samples (97.5%) and 10/10 (100%) fecal samples. The two plasma samples that tested negative with the Altostar assay had a very low HEV RNA concentration (1.6 and 1.4 log10 IU/ml). Quantitative results obtained with the automated platform and the manual workflow were highly correlated (ρ= 0.98, p<0.01). The intra-run and inter-run standard deviation were 0.09 IU/ml and 0.13 IU/ml respectively. The assay was linear from 2 to 6 log IU/ml. The limit of detection determined by Probit analysis with the WHO HEV RNA standard was 7.6 [95% CI: 4.4-52.5] IU/ml. CONCLUSIONS: The Altostar platform enables highly accurate testing for the detection of HEV RNA in stool and the quantification of HEV RNA in plasma. This allowed us to shorten turnaround times and to save time for the technical staff.

Detection of SARS-CoV-2 RNA in a Zoo-Kept Red Fox (Vulpes vulpes).

Many different animal species are susceptible to SARS-CoV-2, including a few Canidae (domestic dog and raccoon dog). So far, only experimental evidence is available concerning SARS-CoV-2 infections in red foxes (Vulpes vulpes). This is the first report of SARS-CoV-2 RNA detection in a sample from a red fox. The RT-qPCR-positive fox was zoo-kept together with another fox and two bears in the Swiss Canton of Zurich. Combined material from a conjunctival and nasal swab collected for canine distemper virus diagnostics tested positive for SARS-CoV-2 RNA with Ct values of 36.9 (E gene assay) and 35.7 (RdRp gene assay). The sample was analysed for SARS-CoV-2 within a research project testing residual routine diagnostic samples from different animal species submitted between spring 2020 and December 2022 to improve knowledge on SARS-CoV-2 infections within different animal species and investigate their potential role in a One Health context. Within this project, 246 samples from 153 different animals from Swiss zoos and other wild animal species all tested SARS-CoV-2 RT-qPCR and/or serologically negative so far, except for the reported fox. The source of SARS-CoV-2 in the fox is unknown. The fox disappeared within the naturally structured enclosure, and the cadaver was not found. No further control measures were undertaken.

Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples.

OBJECTIVES: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. METHODS: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases. RESULTS: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%). DISCUSSION: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries.

Improved SARS-CoV-2 RNA recovery in wastewater matrices using a CTAB-based extraction method.

Wastewater-based epidemiology has allowed tracking the magnitude and distribution of SARS-CoV-2 in communities, allowing public health officials to prepare for impending outbreaks. While many factors influence recovery of SARS-CoV-2 from wastewater, proper extraction, concentration, and purification of RNA are key steps to ensure accurate detection of viral particles. The aim of this study was to compare the efficiency of four commonly used RNA extraction methods for detection of the SARS-CoV-2 RNA genome in sewage samples artificially inoculated with the virus, in order to identify a protocol that improves viral recovery. These methods included CTAB-based, TRIzol-based, and guanidinium thiocyanate (GTC)-based extraction procedures coupled with silica spin column-based purification, and an automated extraction/purification protocol using paramagnetic particles. Following RNA extraction, virus recovery rates were compared using RT-qPCR-based detection. The CTAB-based approach yielded the highest recovery rates and was the only method to consistently demonstrate stable virus recovery percentages regardless of the specific physicochemical characteristics of the samples tested. The TRIzol method proved to be the second most effective, yielding significantly higher recovery rates compared to both the GTC-based and the automated extraction methods. These results suggest that the CTAB-based approach could be a useful tool for the recovery of viral RNA from complex wastewater matrices.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Detection of SARS-CoV-2 RNA in wastewater from an enclosed college campus serves as an early warning surveillance system.

SARS-CoV-2, the causative agent of Covid-19, is shed from infected persons in respiratory droplets, feces, and urine. Using quantitative PCR (qPCR), our group hypothesized that we could detect SARS-CoV-2 in wastewater samples collected on a university campus prior to the detection of the virus in individuals on campus. Wastewater samples were collected 3 times a week from 5 locations on the main campus of the University of North Carolina Wilmington (UNCW) from July 24, 2020 to December 21, 2020. Post-collection, total RNA was extracted and SARS-CoV-2 RNA in the samples was detected by qPCR. SARS-CoV-2 signal was detected on campus beginning on August 19 as classes began and the signal increased in both intensity and breadth as the Fall semester progressed. A comparison of two RNA extraction methods from wastewater showed that SARS-CoV-2 was detected more frequently on filter samples versus the direct extracts. Aligning our wastewater data with the reported SARS-CoV-2 cases on the campus Covid-19 dashboard showed the virus signal was routinely detected in the wastewater prior to clusters of individual cases being reported. These data support the testing of wastewater for the presence of SARS-CoV-2 and may be used as part of a surveillance program for detecting the virus in a community prior to an outbreak occurring and could ultimately be incorporated with other SARS-CoV-2 metrics to better inform public health enabling a quick response to contain or mitigating spread of the virus.

Hepatitis E Virus RNA Detection from Hunted Wild Boars in Central Italy: an Epidemiological Investigation.

Every year, foodborne pathogens, including the hepatitis E virus (HEV), cause thousands of infections in different continents. Final consumers become infected through the ingestion of contaminated animal origin foodstuffs. Generally, in industrialized countries, HEV genotype 3 is involved in sporadic outbreaks. Infections have been described, in Europe and Japan as consequence of pork products and contaminated wild boar's primary or processed products (liver and muscle tissues) consumption. In Central Italy, hunting activities are largely practiced. In these small and rural communities, game meat and liver are ingested by hunters' families or at local and traditional restaurants. Therefore, these food chains can be considered critical HEV reservoirs. In this study, 506 liver and diaphragm tissues were collected from hunted wild boars in the Southern Marche region (Central Italy) and were screened for HEV RNA detection. From the 10.87% of liver and 2.76% of muscle samples, HEV3 subtype c was discovered. The observed prevalence values resulted in line with previous investigations performed in other Central Italian regions, but higher than Northern ones (3.7% and 1.9% from liver tissue). Therefore, the obtained epidemiological data highlighted the wide occurrence of HEV RNA circulation in a low-investigated area. Basing on results, a One-health approach was adopted due to the sanitary relevance of this Public Health concern.

On-Chip Enrichment System for Digital Bioassay Based on Aqueous Two-Phase System.

We developed an on-chip enrichment method based on an aqueous two-phase system of dextran/polyethylene glycol mix, DEX/PEG ATPS, for digital bioassay. Accordingly, we prepared an array device of femtoliter reactors that displays millions of uniformly shaped DEX-rich droplets under a PEG-rich medium. The DEX-rich droplets effectively enriched DNA molecules from the PEG-rich medium. To quantify the enrichment power of the system, we performed a digital bioassay of alkaline phosphatase (ALP). Upon genetically tagging ALP molecules with the DEX-binding domain (DBD) derived from dextransucrase, the ALP molecules were enriched 59-fold in the DEX droplets in comparison to that in a conventional digital bioassay. Subsequently, we performed a Cas13-based digital SARS-CoV-2 RNA detection assay to evaluate the performance of this system for a more practical assay. In this assay, the target RNA molecules bound to the DBD-tagged Cas13 molecules were effectively enriched in the DEX droplets. Consequently, an enrichment factor of 31 was achieved. Enrichment experiments for nonlabeled proteins were also performed to test the expandability of this technique. The model protein, nontagged ß-galactosidase, was enriched in DEX droplets containing DBD-tagged antibody, with an enrichment factor of over 100. Thus, this system enabled effective on-chip enrichment of target molecules to enhance the detection sensitivity of digital bioassays without using external instruments or an external power source, which would be applicable for on-site bioassays or portable diagnostic tests.