Detection Of Coronavirus Research Articles

Feasible Preparation of Naphthalimide-Functionalized Zeolitic Imidazolate Framework-8 for Control, and Fluorescence Detection of Virus: Optimization of Model Study.

The onset of the coronavirus has highlighted the critical need to swift diagnostic techniques to identify the virus. Metal Organic Frameworks (MOFs) have attracted considerable attention in the field of sensing due to the remarkable characteristics. The present research examines the microwave preparation of naphthalimide-functionalized zeolitic imidazolate framework-8 (NI/ZIF-8) to the detection of coronavirus as a probe. The microwave synthesis approach facilitates an environmentally sustainable, and effective production of NI/ZIF-8. Characterization techniques have validated the material's high crystallinity and thermal behaviour. In order to enhance the performance of the probe, a 23 factorial model is implemented. This model facilitates the influences of control parameters, including NI/ZIF-8 dose, solution temperature, and buffer pH, on quenching response. The findings indicate the 27°C, pH 8.2, and a NI/ZIF-8 dose of 0.6mg/mL yields the maximum quenching response (83.21%). Additionally, it has been determined that photo electron transfer plays a significant role within the quenching process. The sensor demonstrated remarkable sensitivity to the COVID-19 RNA, achieving an exceptionally detection limit of 5.45 pM and a quick sense (20min), alongside a high detection selectivity of RNA. This project underscores the applicability of NI/ZIF-8 as an effective and eco-friendly sustainable substance for the rapid and accurate detection of RNA of COVID-19.

Establishment of a Quadruplex RT-qPCR for the Detection of Canine Coronavirus, Canine Respiratory Coronavirus, Canine Adenovirus Type 2, and Canine Norovirus

Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022–2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 102 copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19–1.31% and inter-assay variability of 0.10–0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.

Detection and Prevalence of Coronaviruses in European Bats: A Systematic Review.

Bats are known hosts for a wide range of coronaviruses (CoVs), including those that cause severe acute respiratory syndrome (SARS-CoV-1) and Middle East respiratory syndrome (MERS-CoV). With the emergence of the COVID-19 pandemic caused by the SARS-CoV-2 virus, it has become increasingly important to understand the diversity and prevalence of CoVs in bat populations. This systematic review aimed to compile studies that have sampled CoVs from bats across Europe and assessed various aspects related to the testing of bat samples, including the country where the bats were collected, the CoV genomic region studied, the CoV genera that were detected, and the identification of bat species that were found to be carrying CoV. We identified 30 studies that assessed CoVs presence in bats across multiple countries including Italy, Germany, and various other nations with one or two studies each, which tested them for CoVs using a variety of matrices. CoVs were found in nine genera of bats, and the genomic regions included RdRp, ORF1a gene, as well as full genome, detecting α- and/or β-CoVs, with most of them being detectable only in faeces. This review provides a comprehensive overview of the CoVs detected in bats across Europe and highlights the importance of continued surveillance and monitoring of bat populations for potential emerging zoonotic CoVs.

Self-powered water-based graphene photodetector for extremely rapid detection of SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus, which has caused a serious public health crisis all over the world. Current coronavirus detection methods mainly rely on PCR technology, which needs high standard laboratory facilities and a long time to extract, react and analyze samples. In this report, a self-powered water-based photodetector (PD) biological detection device is designed to detect SARS-CoV-2 in the Dulbecco's Modified Eagle Medium (DMEM) solution. The graphene layer on top of liquid is covered with specific antibodies against SARS-CoV-2 spike protein. The device can detect SARS-CoV-2 exclusively with a concentration of 1 × 104 TCID50 in DMEM solution. It can push the detection limit down to 62 copies per μL with the detection time less than 100 ms. The work presents a rapid SARS-CoV-2 immunodiagnostic method without sample pretreatment or labeling.

Wheat Germ Agglutinin-Modified "Three-in-One" Multifunctional Probe Driven Broad-Spectrum and Flexible Immunochromatographic Diagnosis of viruses With High Sensitivity.

The conventional lateral flow assay (LFA) fails to the demands for the accurate screening of viruses as a result of its low sensitivity of colorimetric signal output and poor universality limited by antibody pairs. Here, a magnetically assisted dual-signal output LFA platform is developed for the ultrasensitive, universal, and flexible detection of viruses. A "three-in-one" multifunctional probe (MAuDQD) is prepared using a 180nm Fe3O4 core to load numerous Au nanoparticles (NPs) and two layers of QDs, which can substantially improve the sensitivity of LFA through coupling with the effects of magnetic enrichment and colorimetric/fluorescent enhancement. Wheat germ agglutinin (WGA)-modified MAuDQD attained the broad-spectrum capture viral membrane proteins and the colorimetric/fluorescent dual-mode detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) on the LFA strip. In the colorimetric mode, the target viruses detected directly, with the visual sensitivity reaching 0.1-0.5ngmL-1 and the fluorescent mode supported quantitative analysis of SARS-CoV-2/MPXV with limits of detection decreasing to pgmL-1 level. Practicability of the MAuDQD@WGA-LFA is verified through the detection of 33 real clinical samples, showing the proposed assay has a great potential to become a sensitive, accurate, and universal tool for on-site monitoring of viral infections.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

Simulation of fractional order mathematical model of robots for detection of coronavirus using Levenberg–Marquardt backpropagation neural network

Simulation of fractional order mathematical model of robots for detection of coronavirus using Levenberg–Marquardt backpropagation neural network

Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a Micro-Air-Filtration Device Format.

The Coronavirus disease 2019 (COVID-19) pandemic has supercharged innovation in the field of molecular diagnostics and led to the exploration of systems that permit the autonomous identification of airborne infectious agents. Airborne virus detection is an emerging approach for determining exposure risk, although current methods limit intervention timeliness. Here, we explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for one-pot detection of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters suitable for micro-air-filtration of airborne viruses. We use a design of experiments statistical framework to establish the optimal additive composition for running RT-LAMP on membrane filters. Using SCV2 liquid spike-in experiments and fluorescence detection, we show that single-pot RT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture infectious dose (TCID50) SCV2 per reaction (3600 E-gene copies) and is an order of magnitude more sensitive than conventional RT-LAMP.

Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19–29aa, RMSD 1–1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Advancing SARS‐CoV‐2 Variant Detection with High Affinity Monoclonal Antibodies and Plasmonic Nanostructure

AbstractA nanoplasmonic biosensor for the precise detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by leveraging advances in nanostructured sensing materials and highly selective monoclonal antibodies is presented. The sensor integrates plasmonic Au nanostructures optimized for surface‐enhanced Raman scattering (SERS), enhancing sensitivity through unique light interactions at the nanoscale. Coupled with exclusive antibodies that specifically target SARS‐CoV‐2 and its evolving variants, this sensor demonstrates remarkable selectivity and versatility. Validated with 270 clinical samples, it demonstrates a sensitivity of 98.9% and specificity of 100%. More importantly, the virus in nasopharyngeal swab samples collected over 3 years, marking the long‐term, large‐scale clinical validation of the nanoplasmonic biosensor for SARS‐CoV‐2 is been successfully detected. Furthermore, the integration of this sensor with face masks enables the detection of airborne SARS‐CoV‐2, highlighting its potential for routine management of coronavirus disease 2019 (COVID‐19).

Development and Diagnosis Performance of IgM-Based Rapid Antigen Test for Early Detection of SARS-CoV-2 Infection in a Large Cohort of Suspected COVID-19 Cases - USA, Poland, and Sweden, 2021-2022.

Antigen testing has been crucial in effectively managing the coronavirus disease 2019 (COVID-19) pandemic. This study evaluated the clinical performance of a nasopharyngeal swab (NPS)-based antigen rapid diagnostic test (Ag-RDT) compared to the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) for early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We developed an IgM-based rapid antigen test for early detection of SARS-CoV-2 infection. Between July 2021 and January 2022, we analyzed 1,030 NPS samples from participants at three centers in different countries, using both antigen rapid diagnostic tests (Ag-RDT) and RT-PCR. The Ag-RDT demonstrated minimal detection limits as low as 0.1 ng/mL for recombinant N antigen and 100 TCID50/mL for heat-inactivated SARS-CoV-2 virus. Specificity assessments involving four human coronaviruses and 13 other respiratory viruses showed no cross-reactivity. The Ag-RDT assay (ALLtest) exhibited high sensitivity (93.18%-100%) and specificity (99.67%-100%) across all centers. Factors such as cycle threshold (Ct) values and the timing of symptoms since onset were influential, with sensitivity increasing at lower Ct values (<30) and within the first week of symptoms. The ALLtest Ag-RDT demonstrated high reliability and significant potential for diagnosing suspected COVID-19 cases.

The Role of Biosensors in Detection of SARS-CoV-2: State-of-the-Art and Future Prospects

Abstract: The world is fighting a pandemic so grave that perhaps it has never been witnessed before; COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). As of August 31st, 2022, the WHO declared the total number of confirmed cases was 599,825,400, with 6,469,458 confirmed deaths from 223 countries under the scourge of this deadly virus. The SARS-CoV-2 is a β-coronavirus, which is an enveloped non-segmented positive- sense RNA virus. It is a close relative of the SARS and MERS viruses and has probably entered humans through bats. Human-to-human transmission is very rapid. People in contact with the patient or even the carriers became infected, leading to a widespread chain of contamination. We are presenting a mini-review on the role of biosensors in detecting SARS-CoV-2. Biosensors have been used for a very long time for viral detection and can be utilized for the prompt detection of the novel coronavirus. This article aims to provide a mini-review on the application of biosensors for the detection of the novel coronavirus with a focus on costeffective paper-based sensors, nanobiosensors, Field effect transistors (FETs), and lab-on-chip integrated platforms. Background: Biosensors have played a crucial role in viral detection for a long time. Objective: To present a comprehensive review of the biosensor application in SARS-Cov-2. Method: We have presented state-of-the-art work in the biosensors field for SARS-Cov-2 detection. Results: The biosensors presented here provide an innovative approach to detecting SARS-Cov- 2 infections early. Conclusion: Biosensors have tremendous potential in accurately detecting viral infections in pandemics requiring rapid screening.

Utility of Extraction-Free SARS-CoV-2 Detection by RT-qPCR for COVID-19 Testing in a Resource-Limited Setting.

The COVID-19 epidemic had a profound impact on global health and the economy and Ghana was no exception to its far-reaching consequences. Regarding detection of the causative agent-the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse-transcription-qPCR (RT-qPCR) is widely recognized as a very sensitive and reliable diagnostic technique used globally. There are, however, high operational costs in acquiring test kits, equipment, and accessories for RT-qPCR testing, which pose significant challenges in resource-limited settings. Hence, this proof-of-concept study set out to develop a more affordable COVID-19 protocol for use in low or lower-middle-income settings, such as Ghana, that would bypass the traditional extraction process using inexpensive reagents and evaluate the possibility of processing samples collected using wooden shaft swabs. Several less expensive media were used for the extraction-free process. Results demonstrated that direct RT-qPCR assay after 5 min heat inactivation of virus at 95 °C in 0.1× PBS or molecular grade water resulted in viral detection with quantification cycle (Cq) values that are comparable to results obtained following the extraction process. Also, wooden shaft swabs could be used for sampling if incubation times are kept to less than 6 h. The study demonstrates that extraction-free protocols are one way to minimize the cost of COVID-19 testing by RT-qPCR.

Simple and Ultrasensitive Nanozyme-Linked Immunosorbent Assay for SARS-CoV-2 Detection on a Syringe-Driven Filtration Device.

This work proposed a simple and ultrasensitive nanozyme-based immunoassay on a filtration device for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP). Gold core porous platinum shell nanoparticles (Au@Pt NPs) were synthesized with high catalytic activity to oxidize 3,3',5,5'-tetramethylbenzidine, leading to an oblivious color change. The filtration device was designed based on the size difference of magnetic beads, filter membrane pore, and Au@Pt NPs. A simple, rapid, and consistent washing procedure can be performed with the help of a plastic syringe. This detection method could realize the quantitative detection of SARS-CoV-2 NP within 80 min for point-of-care needs. The limit of detection for the SARS-CoV-2 antigen was 0.01 ng/mL in buffer. The coefficients of variation of the assay were 1.78% for 10 ng/mL SARS-CoV-2 antigen, 2.03% for 1 ng/mL SARS-CoV-2 antigen, and 2.34% for the negative sample, respectively. The specificity of the detection platform was verified by the detection of various respiratory viruses. This simple and effective detection system was expected to promote substantial progress in the development and application of virus immunodetection technology.

Diagnostic accuracy of Savanna RVP4 (QuidelOrtho) for the detection of Influenza A virus, RSV, and SARS-CoV-2.

We assessed the diagnostic accuracy of a new point-of-care test for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV). The new test has a concordance with the reference standard of 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.1% (RSV). The sensitivity of 85%-95% and specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV is comparable with similar nucleic acid amplification-based point of care tests but at lower costs.

Applications of nanostructured materials for severe acute respiratory syndrome-CoV-2 diagnostic

There is a growing concern that severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infections will continue to rise, and there is now no safe and effective vaccination available to prevent a pandemic. This has increased the need for rapid, sensitive, and highly selective diagnostic techniques for coronavirus disease (COVID-19) detection to levels never seen before. Researchers are now looking at other biosensing techniques that may be able to detect the COVID-19 infection and stop its spread. According to high sensitivity, and selectivity that could provide real-time results at a reasonable cost, nanomaterial show great promise for quick coronavirus detection. In order to better comprehend the rapid course of the infection and administer more effective treatments, these diagnostic methods can be used for widespread COVID-19 identification. This article summarises the current state of research into nanomaterial-based biosensors for quick SARS‑CoV‑2 diagnosis as well as the prospects for future advancement in this field. This research will be very useful during the COVID-19 epidemic in terms of establishing rules for designing nanostructure materials to deal with the outbreak. In order to predict the spread of the SARS-CoV-2 virus, we investigate the advantages of using nano-structure material and its biosensing applications.

Validating Combination Throat-Nasal Swab Specimens for COVID-19 Tests Would Improve Early Detection, Especially for the Most Vulnerable.

Early detection of severe acute respiratory syndrome coronavirus 2 infection by diagnostic tests can prompt actions to reduce transmission and improve treatment efficacy, especially for vulnerable groups such as immunocompromised individuals. Recent evidence suggests that sampling the throat in addition to the nose improves clinical sensitivity during early infection for both antigen and molecular coronavirus disease 2019 tests. We urge test manufacturers to validate tests for use with throat swab, in combination with nasal swabs.

Upconversion luminescence resonance energy transfer (LRET)-based dual-channel biosensor for rapid detection of coronavirus

Researchers recently have been working on a sensitive, accurate, and easily accessible way to detect coronaviruses. Traditional polymerase chain reaction detection technology has high sensitivity and specificity, but there are some problems, such as a complicated detection process, false negatives caused by gene mutation of coronavirus, and easy infection of operators. Antigen detection, which does not require gene amplification and other preprocessing, can be operated by non-medical staff for rapid detection. However, its detection sensitivity requires high reliability and further improvement. Accordingly, a biosensor based on luminescent resonance energy transfer (LRET) from upconversion nanoparticles (UCNPs) to gold nanoparticles (AuNPs) and antigen detection has been developed for rapid and sensitive detection of N protein of SARS-CoV-2. By adjusting the reaction conditions, LRET can be realized by making the spectral overlap between the luminescence of UCNPs and the absorption band of AuNPs, which can effectively avoid the generation of biological background light and the interference of other signals in the test process. In addition, the thiol-ene click reaction was used to link Thiol-PEG-COOH to UCNPs to improve its biocompatibility. The antigen recognition of the coronavirus based on the as-proposed detection method can be further realized on the test paper. A linear response is obtained ranging from 10 ng mL−1 to 1280 ng mL−1, and the detection limit is around 0.80 ng mL−1 with a detection time of around 10 minutes, which has a wide range of potential future. This fast and sensitive biosensor for coronavirus detection can be further reflected in the test paper and is promising for practical applications.

External Quality Assessment Program for SARS-COV-2 Molecular Detection in Pakistan.

Amid coronavirus disease 2019 (COVID-19) pandemic, accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for diagnosis management and breaking down transmission chains. We designed a national external quality assessment panel (EQAP) for SARS-CoV-2 molecular detection comprising working laboratories nationwide. A molecular diagnostic EQA panel that consists of five samples for SARS CoV-2 testing was distributed to 141 public and private sector laboratories across country. These samples contain different concentrations of SARS-CoV-2 to evaluate the sensitivity of commercial kits available. Sensitivity among public and private sector laboratories was variable, particularly lower SARS-CoV-2 concentrations significantly increased the risk of false-negative tests, whereas Ct values of accurately tested SARS-CoV-2 specimens increased as concentration decreased. These findings highlighted that performance of used commercial kits was not significantly correlated to various extraction or PCR methods. This study highlights the need for a national external quality assessment panel (EQAP) in the country to improve the quality of the healthcare system while ensuring the accuracy and reliability of results. Furthermore, EQAPs can help laboratories meet accreditation and regulatory requirements. However, continued participation in EQAP is recommended for quality enhancement of laboratories.

Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.

Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.