Development of a platform for in-situ airborne virus detection by interfacing RT-LAMP assay with a condensational growth collector

Integration of sample preparation with RNA-Amplification in a hand-held device for airborne virus detection

Foot and mouth disease is infectious, tranboundary, disease of cloven hoofed domesticated ruminants. In this study, ELISA confirmed positive infected and field samples were analyzed through RT-PCR using universal primer set (IF/IR and P1/P2) and RT-LAMP assays. A total of N=100 samples were collected from different livestock population areas in Punjab. Types of samples were serum, nasal secretions, tissue, saliva and swab samples. Out of N=100 infected samples, n=19 samples were serotyped into A, O and Asia 1 strains through antigen ELISA (Ag- ELISA) and these samples were further confirmed through RT-PCR and RT-LAMP assays. ELISA confirmed samples revealed 100% results with RT-PCR and RT-LAMP assays. Field samples revealed 33.33%, 27.16% and 37.03% with RT-PCR (IF/IR and P1/P2) and RT-LAMP assays respectively. It was concluded that ELISA confirmed samples were successfully analyzed with RT-PCR and RT-LAMP assays. Both the assays successfully analyzed 2017 outbreaks of FMD in Punjab.

Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these ‘look-a-like’ diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV’s and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.

Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses

The present study was undertaken to see the excretion of FMD virus in milk during and after the subset of FMD outbreak. Fore-milk (50 ml) was sampled from 12 clinically infected and 3 asymptomatic cows in the morning. Analytical sensitivity of NAR methods was estimated using uninfected negative milk sample spiked with 105.7 TCID50/ml FMD serotype O virus (IND R2/1975) in 10 fold serial dilution. Detection limit of mPCR and RT- LAMP assay was 102.7 and 101.7 TCID50/ml, respectively. 15 individual and pooled cows’ milk samples infected with FMD virus were processed for virus isolation (VI) and detection till 37 days post clinical manifestation (dpm). Virus isolation from individual and pooled milk from infected cow was positive till 6 and 4 dpm, respectively. Individual milk and pooled milk samples were found positive by m-PCR till 37 and 14 dpm, respectively, but by RT-LAMP till 37 and 21 dpm, respectively. In case of asymptomatic cows, viral genome was detected 2–5 days before appearance of disease in other animals. Milk virus isolate had 100% nucleotide identity at VP1 coding region. mPCR and RT LAMP assays has potential to detect FMD virus in milk and help to prevent the spread of FMD virus from one place to another place.

Viruses offer a limited range of targets for their detection. To date, PCR and RT-PCR have been widely used for detection of viruses. In the case of environmental air sampling, the ability to detect a broad range of viruses would constitute a significant advantage for preventing outbreaks of airborne-transmitted viral infections. Given that neuraminidase is found on some respiratory virus species of medical or agricultural importance, this enzyme could theoretically be used to detect several different airborne viruses in a single assay. The aim of the present study was to evaluate the potential of neuraminidase activity as a marker for rapid detection of airborne viruses. We first validated the use of a low-pathogenic strain of Newcastle disease virus (NDV) as a model airborne virus. Our findings revealed that neuraminidase activity-based assays are almost as sensitive as RT-PCR assays currently used for detection of NDV. We also validated the utilization of a neuraminidase substrate specific to viral neu...

: Airborne virus outbreaks, including the influenza pandemic of 1918, the recent SARS pandemic and the anticipated H5N1 outbreak, plus the perceived threat of bioterrorism warrant concern about the prevalence and potential effects of airborne viruses. However, current bioaerosol sampling methods do not effectively sample airborne viruses (typically 20 - 300 nm). To address this problem, a novel Bioaerosol Amplification Unit (BAU) has been designed and constructed to increase the size of the virus particles by condensational growth, thereby enhancing sample recovery. In this study reaerosolization of viral particles from the impinger was investigated to assess its impact on the capability of the BAU. Reaerosolization was characterized as a function of flow rate and concentration of the collection liquid in the impinger. An impinger containing a known concentration of particles (MS2 bacteriophage or polystyrene latex) was operated at various flow rates with sterile air, and a scanning mobility particle sizer was used to determine the reaerosolization rates. Results indicate that reaerosolization increased with increasing flow rate due to the additional energy added to the system. However, reaerosolization increased with concentration up to ~10e4 PFU/mL and then decreased at progressively higher concentrations. This phenomenon likely resulted from aggregation of viral particles or increased surface tension or viscosity at higher concentration. Adjusting surface tension by adding soap and increasing viscosity by adding a layer of heavy white mineral oil decreased reaerosolization. Thus, reaerosolization from an impinger could compromise the improved collection capability of the BAU

Due to the highly contagious and spreads quickly of respiratory infectious diseases (ADR), the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Here, we develop an approach based on loop-mediated isothermal amplification (LAMP) for the detection of influenza A virus (Flu A), Flu A subtypes H1N1and H3N2, influenza B virus (Flu B), respiratory syncytial virus (RSV) subtypes A and B, human adenovirus (HAdV), parainfluenza virus (PIV) subtypes 1 and 3, and human rhinovirus (HRV) simultaneously. We designed primers specific to detect respiratory viruses above, optimized the RT-LAMP assay and evaluated it for its sensitivity and specificity of detection using real-time monitoring based on SYBR Green I. We also evaluated the result of our RT-LAMP assay on 638 nasopharyngeal swab specimens with the commercial RT-PCR by Cohen’s Kappa. The inconsistent results were verified by Sanger sequencing furtherly. The developed RT-LAMP assay displayed a detection limit of 1 × 102 copies/ml RNA close to that of RT-PCR; no cross-reactivity was observed in the 10 kinds of viruses studied. The results obtained with 638 clinical samples indicate that the developed method has high specificity (0.988–1) and sensitivity (0.863–1) for viruses studied, and the Kappa value of all viruses was more than 0.85 revealed an excellent agreement between the two methods. We developed an RT-LAMP-based method and optimized for the detection of common respiratory viruses. It may be a powerful tool for rapid and reliable clinical diagnosis of ADR in primary hospitals.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10096-021-04300-8.

A new duck Tembusu virus (TMUV), also known as BYD virus, has been identified as the causative agent for the emerging duck egg-drop syndrome in mainland China. The rapid spread and wide distribution of the new TMUV in mainland China result in heavy loss to the poultry industry and pose great threats to public health. Rapid and sensitive detection methods are critical for prevention and control of TMUV infections. In this study, a reverse-transcription loop-mediated isothermal amplification assay (RT-LAMP) and an SYBR Green-I-based real-time RT-PCR assay specific for the duck TMUV were developed and validated with laboratory and field samples, respectively. The detection limits were 1 × 10(-4) and 1 × 10(-3) PFU per reaction for the RT-LAMP assay and real-time RT-PCR assay, respectively. The specificities were analyzed with other related members of the genus Flavivirus, and no cross-reaction was observed. Furthermore, both assays were evaluated with field samples, and they exhibited high sensitivity and specificity. In addition, the real-time RT-PCR assay worked well in viral load analysis, which revealed that the spleen may be the primary target for the replication of new duck TMUV in ducks. The TMUV-specific RT-LAMP and real-time RT-PCR assays will provide useful tools for the diagnosis and epidemiological surveillance of TMUV infection.

A simple colorimetric detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification assay using hydroxynaphthol blue metal indicator

There is an urgent need for efficient airborne virus monitoring to proactively cut off transmission sources in the public. However, infected respiratory sprays can remain stable in the airborne aerosols, and the sampling of airborne viruses has low efficiency. In this work, inspired by the cactus conical leaves and redbud venation, we propose an approach for the capture of airborne virus and in situ detection. A paraffin kirigami with cactus cone-shaped spine units arranged to mimic leaf veins was designed to integrate with electrochemical monitoring as an origami card. With the combined effect of Laplace pressure, gravity assistance, leaf vein-mimicking structure, and aerodynamic vortices induced by the assembly of the origami card, the paraffin kirigami card demonstrates excellent ability to collect virus-containing sprays from air and facilitate directional transport and aggregation. The peak fog harvesting rate reaches 5424.2 mg/cm2/h, demonstrating exceptional efficiency in atmospheric droplet capture. A triad electrode system is accommodated at the root of the spray collector to monitor airborne virus in the collected droplets. The paraffin kirigami card achieves a low limit of detection (LOD) of 0.10776 copies/mL and shows a good linear relationship between the impedance and influenza A (H1N1) virus concentration. Validation using thrombin as a model target analyte confirmed the system's integrated capability for rapid collection, identification, and sensitive detection of viruses. This cactus-inspired paraffin kirigami card demonstrates significant potential for real-time, in situ airborne virus collection and detection, contributing to efforts to control the spread of viruses.

Highly sensitive airborne virus monitoring is critical for preventing and containing epidemics. However, the detection of airborne viruses at ultra-low concentrations remains challenging due to the lack of ultra-sensitive methods and easy-to-deployment equipment. Here, we present an integrated microfluidic cartridge that can accurately detect SARS-COV-2, Influenza A, B, and respiratory syncytial virus with a sensitivity of 10 copies/mL. When integrated with a high-flow aerosol sampler, our microdevice can achieve a sub-single-copy spatial resolution of 0.83 copies/m3 for airborne virus surveillance with an air flow rate of 400 L/min and a sampling time of 30 minutes. We then designed a series of virus-in-aerosols monitoring systems (RIAMs), including versions of a multi-site sampling RIAMs (M-RIAMs), a stationary real-time RIAMs (S-RIAMs), and a roaming real-time RIAMs (R-RIAMs) for different application scenarios. Using M-RIAMs, we performed a comprehensive evaluation of 210 environmental samples from COVID-19 patient wards, including 30 aerosol samples. The highest positive detection rate of aerosol samples (60%) proved the aerosol-based SARS-CoV-2 monitoring represents an effective method for spatial risk assessment. The detection of 78 aerosol samples in real-world settings via S-RIAMs confirmed its reliability for ultra-sensitive and continuous airborne virus monitoring. Therefore, RIAMs shows the potential as an effective solution for mitigating the risk of airborne virus transmission.

A squash print reverse transcription loop-mediated isothermal amplification amplification (SP-RT-LAMP) assay was developed for detection of Potato leafroll virus (PLRV) in a single aphid. Healthy aphids were given an acquisition feeding on potato plants infected with PLRV, and the acquired aphids were used for RNA isolation. The RT-LAMP assay was carried out using LAMP primers targeting the coat protein gene of PLRV. The amplified product was run on agarose gel; a typical ladder-like pattern in the acquired aphids indicated that the assay was able to detect PLRV from an individual aphid. The results of the RT-LAMP assay for the detection of PLRV in a single aphid were also visualized directly by adding fluorescent dye in LAMP amplified product. These results were confirmed by RT-PCR detection where an amplicon of expected size of 492 bp was observed in virus-acquired single aphids. The assay was optimized with respect to its isothermal amplification temperature and its incubation period. The technique was examined for its relative selectivity with other potato viruses transmitted by aphids where it did not show any cross-reactivity with the tested viruses indicating its specificity. With respect to its relative sensitivity, it was found to be equally sensitive as the existing RT-PCR assay. It was also found to be robust and highly reproducible as it was able to detect PLRV from known and unknown single aphid samples. The assay was successful in detection of PLRV in potato plants and tubers as well. Hence, we conclude that the assay is simple, sensitive and economical for detection of PLRV in single aphid and in potato plants and tubers.

Tomato mosaic virus (ToMV), an economically important virus that affects a wide range of crops, is highly contagious, and its transmission is mediated by mechanical means, and through contaminated seeds or planting materials, making its management challenging. To contain its wide distribution, early and accurate detection of infection is required. A survey was conducted between January and May, 2023 in major tomato growing counties in Kenya, namely, Baringo, Kajiado, Kirinyaga and Laikipia, to establish ToMV disease incidence and to collect samples for optimization of the reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay. A RT-LAMP assay, utilizing primers targeting the coat protein, was developed and evaluated for its performance. The method was able to detect ToMV in tomato samples within 4:45 minutes, had a 1,000-fold higher sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) method and was specific to ToMV. Furthermore, the practical applicability of the assay was assessed using tomato samples and other solanaecous plants. The assay was able to detect the virus in 14 tomato leaf samples collected from the field, compared to 11 samples detected by RT-PCR, further supporting the greater sensitivity of the assay. To make the assay more amenable for on-site ToMV detection, a quick-extraction method based on alkaline polyethylene glycol buffer was evaluated, which permitted the direct detection of the target virus from crude leaf extracts. Due to its high sensitivity, specificity and rapidity, the RT-LAMP method could be valuable for field surveys and quarantine inspections towards a robust management of ToMV infections.

One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of Cymbidium mosaic virus