Viruses In Clinical Samples Research Articles

An electrochemical, fluorescent, and colorimetric triple-mode homogeneous biosensor for the simultaneous detection of influenza A, influenza B, and SARS-CoV-2

Influenza A, influenza B, and SARS-CoV-2 are prevalent during the influenza season, and their symptoms are very similar. To enhance symptomatic and precise treatment, there is an urgent need for multi-pathogen simultaneous detection technology to rapidly identify the three viruses. Therefore, a homogeneous nucleic acid detection system has been developed based on DNA-RNA hybridization. This system depends on the most crucial triple-mode probe consisting of a common fluorophore and an electrochemically active group to produce electrochemical, fluorescent, and colorimetric signal responses. Based on the electrochemical signal, supplemented by fluorescent and colorimetric signals, it enables rapid and sensitive detection of influenza A, influenza B, and SARS-CoV-2, with a detection limit of 0.3 aM for synthetic DNA in 40 minutes, and a detection limit of 100 CFU/mL for engineered bacteria. In simulated sample detection, the system enables accurate identification of samples from multiple sources, such as serum, saliva, and nasopharyngeal swab, with a wide sample applicability range. In addition, the system can be utilized in conjunction with a microfluidic chip and a portable electrochemical instrument to simultaneously detect three viruses in clinical samples, making it convenient for non-professionals in remote areas to obtain results in a short period of time. This work provides a simple and scalable research idea and solution for the simultaneous detection of pathogens.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

Comprehensive analysis of antigenic variations and genomic properties of hepatitis B virus in clinical samples in the mid-north east region of Bangladesh

This investigation delineates an exhaustive analysis of the clinical, immunological, and genomic landscapes of hepatitis B virus (HBV) infection across a cohort of 22 verified patients. The demographic analysis unveiled a pronounced male bias (77.27%), with patient ages spanning 20 to 85 years and durations of illness ranging from 10 days to 4 years. Predominant clinical manifestations included fever, fatigue, anorexia, abdominal discomfort, and arthralgia, alongside observed co-morbidities such as chronic renal disorders and hepatocellular carcinoma. Antigenic profiling of the HBV envelope proteins elucidated significant heterogeneity among the infected subjects, particularly highlighted by discordances in the detection capabilities of small and large HBsAg assays, suggesting antigenic diversity. Quantitative assessment of viral loads unveiled a broad spectrum, accompanied by atypical HBeAg reactivity patterns, challenging the reliability of existing serological markers. Correlative studies between viral burden and antigenicity of the envelope proteins unearthed phenomena indicative of diagnostic evasion. Notably, samples demonstrating robust viral replication were paradoxically undetectable by the large HBsAg ELISA kit, advocating for more sophisticated diagnostic methodologies. Genotypic examination of three HBV isolates classified them as genotype D (D2), with phylogenetic alignment to strains from various global origins. Mutational profiling identified pivotal mutations within the basic core promoter and preS2/S1 regions, associated with an augmented risk of hepatocellular carcinoma. Further, mutations discerned in the small HBsAg and RT/overlap regions were recognized as contributors to vaccine and/or diagnostic escape mechanisms. In summation, this scholarly discourse elucidates the intricate interplay of clinical presentations, antigenic diversity, and genomic attributes in HBV infection, accentuating the imperative for ongoing investigative endeavors to refine diagnostic and therapeutic modalities.

Fluorogenic RNA aptamer output sensors via transcription activated by recombinase polymerase amplification for nucleic acid testing

Sensitive, simple, and rapid nucleic acid testing used for pathogen detection are crucial for preventing outbreaks and transmission of infectious diseases. In this study, we present a fluorogenic RNA aptamer Output Sensors via transcription Activated by Recombinase polymerase amplification (RPA), termed ROSAR, for highly sensitive detection of DNA and RNA from pathogenic bacteria or viruses in clinical samples. The promoter primer and reporter primer facilitate the integration of the transcriptional promoter with the fluorogenic RNA aptamer transcription templates through the RPA of target DNA. Subsequently, the amplification products are transcribed by T7 RNA polymerase, producing RNA aptamer that binds to a fluorogenic dye. The assay, which can be completed within 50 min and can allow a limit of detection of level aM nucleic acid concentration, owning to the dual amplification effects of RPA and in vitro transcription. Furthermore, we develop a paper-based ROSAR by freeze-drying the reaction components, which is compatible with visual detection based on mobile phone fluorescent reader and suitable for point-of-care (POC) applications. The assay can detect various viral and bacterial nucleic acids, making it a highly promising tool for large-scale epidemic screening and rapid POC testing in home healthcare settings.

Modified transport medium for improving influenza virus detection.

Accurate detection of influenza virus in clinical samples requires correct execution of all aspects of the detection test. If the viral load in a sample is below the detection limit, a false negative result may be obtained. To overcome this issue, we developed a modified transport medium (MTM) for clinical sample transportation to increase viral detection sensitivity. We first validated the MTM using laboratory-stocked influenza A viruses (IAVs: H1N1, H3N2, H7N3, H9N2) and influenza B viruses (IBVs: Yamagata, Victoria). We also tested clinical samples. A total of 110 patients were enrolled and a pair of samples were collected to determine the sensitivity of real-time polymerase chain reaction (RT-PCR) following MTM treatment. After 24 h culturing in MTM, the viral loads were increased, represented by a 10-fold increase in detection sensitivity for H1N1, H9N2, and IBVs, a 100-fold increase for H3N2, and a 1,000-fold increase for H7N3. We further tested the effects of MTM on 19 IAV and 11 IBV stored clinical samples. The RT-PCR results showed that the positive detection rate of IAV samples increased from 63.16% (12/19) without MTM culturing to 78.95% (15/19) after 48 h culturing, and finally 89.47% (17/19) after 72 h culturing. MTM treatment of IBV clinical samples also increased the positive detection rate from 36.36% (4/11, 0 h) to 63.64% (7/11, 48 h) to 72.73% (8/11, 72 h). For clinical samples detected by RT-PCR, MTM outperformed other transport mediums in terms of viral detection rate (11.81% increase, P=0.007). Our results demonstrated that the use of MTM for clinical applications can increase detection sensitivity, thus facilitating the accurate diagnosis of influenza infection.

Clinical applications of spectroscopic techniques in conjunction with multivariate analysis in virus diagnosis

In view of the global pandemic that started in 2020, caused by COVID-19, the importance of the existence of fast, reliable, cheap diagnostic techniques capable of detecting the virus even in the first days of infection became evident. This review discusses studies involving the use of spectroscopic techniques in the detection of viruses in clinical samples. Techniques based on mid-infrared, near-infrared, Raman, and molecular fluorescence are explained and it was demonstrated how they can be used in conjunction with computational tools of multivariate analysis to build models capable of detecting viruses. Studies that used real clinical samples from 2011 to 2021 were analyzed. The results demonstrate the potential of the techniques in detecting viruses. Spectroscopic techniques, as well as chemometric techniques, were also explained. Viral diagnosis based on spectroscopy has interesting advantages compared to standard techniques such as: fast results, no need for reagents, non-destructiveness for the sample, no need for sample preparation, relatively low cost, among others. Several studies have corroborated the real possibility that, in the near future, we may have spectroscopic tools being successfully applied in viral diagnosis.

Normalization of RT-PCR protocol for detecting dengue viral RNA in infected clinical residual serum samples

Among mosquito-borne viral infections, dengue tops the list with a high prevalence of all four serotypes, especially in tropical regions. The disease has disseminated in over 100 countries and has become a global threat, mainly affecting young individuals. Detecting the dengue virus in clinical samples has a significant role in diagnosing, treating, epidemiology, surveillance, predicting outbreaks etc. To accomplish this, RT-PCR technology has become a practical way out. Although PCR has reached its cutting edge with its improved versions being available, the conventional form of it has still retained the standard for generating more accurate and reliable results cost-effectively. In the present study, the conventional RT-PCR protocol was normalized for detecting dengue viral RNA in infected clinical residual serum samples by adopting a few previous protocols with required modifications. This procedure, with necessary precautions considered particularly for residual samples, resulted in obtaining expected amplicon bands in three samples. Additionally, Sanger sequencing of the amplicons and NCBI nucleotide BLAST analysis of obtained sequences assured the RT-PCR outcomes. The benefits of using clinical residual samples in dengue research encouraged us to make the PCR protocol suitable for such samples by tackling their limitations.

Technical comparison of MinIon and Illumina technologies for genotyping Chikungunya virus in clinical samples

New-generation sequencing (NGS) techniques have brought the opportunity for genomic monitoring of several microorganisms potentially relevant to public health. The establishment of different methods with different mechanisms provides a wide choice, taking into account several aspects. With that in mind, the present aim of the study was to compare basic genomic sequencing metrics that could potentially impact genotyping by nanopores from Oxford Nanopore Technologies and by synthesis from Illumina in clinical samples positive for Chikungunya (CHIKV). Among the metrics studied, running time, read production, and Q score were better represented in Illumina sequencing, while the MinIOn platform showed better response time and greater diversity of generated files. That said, it was possible to establish differences between the studied metrics in addition to verifying that the distinctions in the methods did not impact the identification of the CHIKV virus genotype.

Performance of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Targeting the RNA Polymerase Gene for the Direct Detection of SARS-CoV2 in Nasopharyngeal Swabs.

In 2020, a global pandemic caused by SARS-CoV-2 was declared. Different institutes proposed diagnostic molecular methods to detect the virus in clinical samples. This study aims to validate and standardize the use of a loop-mediated isothermal amplification (LAMP)-based methodology targeting the viral RP gene, as a faster and low-cost diagnostic method for SARS-CoV-2 infections. The results obtained with RT-LAMP (Reverse Transcriptase) were compared to the results of real-time polymerase chain reaction (RT-PCR) to assess its sensitivity and specificity. In total, 115 samples (nasopharyngeal samples) were used for detecting SARS-CoV-2 by RT-LAMP, with 43 positives and 72 negatives. The study showed a positive predictive value (PPV) of 90.7% and a negative predictive value (VPN) of 100%. The LAMP assay also demonstrated a high sensitivity of 90.7% and a specificity of 100% (confidence interval 77.9-97.4%) when using the lower detection limit of 40 copies/µL. The RT-LAMP described here has the potential to detect even the new variants of SARS-CoV-2, suggesting that it may not be significantly affected by gene mutations. The RT-LAMP targeting the RP viral region is faster and less expensive than other molecular approaches, making it an alternative for developing countries.

Proteome2virus: Shotgun mass spectrometry data analysis pipeline for virus identification

ObjectivesShotgun proteomics is a generic method enabling detection of multiple viral species in one assay. The reliable and accurate identification of these viral species by analyzing peptides from MS-spectra is a challenging task. The aim of this study was to develop an easy accessible proteome analysis approach for the identification of viruses that cause respiratory and gastrointestinal infections. MethodsFor this purpose, a shotgun proteomics based method and a web application, ‘proteome2virus’, were developed. Identified peptides were searched in a database comprising proteomic data of 46 viruses known to be infectious to humans. ResultsThe method was successfully tested for cultured viruses and eight fecal samples consisting of ten different viral species from seven different virus families, including SARS-CoV-2. The samples were prepared with two different sample preparation methods and were measured with two different mass spectrometers. ConclusionsThe results demonstrate that the developed web application is applicable to different MS data sets, generated from two different instruments, and that with this approach a high variety of clinically relevant viral species can be identified. This emphasizes the potential and feasibility for the diagnosis of a wide range of viruses in clinical samples with a single shotgun proteomics analysis.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore.

A digital platform that can rapidly and accurately diagnose pathogenic viral variants, including SARS-CoV-2, will minimize pandemics, public anxiety, and economic losses. We recently reported an artificial intelligence (AI)-nanopore platform that enables testing for Wuhan SARS-CoV-2 with high sensitivity and specificity within five minutes. However, which parts of the virus are recognized by the platform are unknown. Similarly, whether the platform can detect SARS-CoV-2 variants or the presence of the virus in clinical samples needs further study. Here, we demonstrated the platform can distinguish SARS-CoV-2 variants. Further, it identified mutated Wuhan SARS-CoV-2 expressing spike proteins of the delta and omicron variants, indicating it discriminates spike proteins. Finally, we used the platform to identify omicron variants with a sensitivity and specificity of 100% and 94%, respectively, in saliva specimens from COVID-19 patients. Thus, our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants.

A novel colorimetric biosensor for rapid detection of dengue virus upon acid-induced aggregation of colloidal gold.

The dengue virus, once transmitted to people through a mosquito bite, causes an infectious disease called dengue fever. Dengue fever can develop into two fatal syndromes, namely dengue shock syndrome and dengue hemorrhagic fever. The existing strategies for detecting dengue infection mainly employ serological immunoassays and a real time PCR technique. Along with the positive features of efficiency, accuracy, and reproducibility, these procedures are limited by being time-consuming, costly, requiring special equipment for analysis, and unable to be carried out at the point-of-care level. Herein, we developed a colorimetric nanosensor for detecting dengue virus in clinical samples that is rapid, accurate, sensitive, and less expensive. The sensing platform relies on the specific binding between the DNA-conjugated AuNPs and genomic RNA of dengue, which results in the DNA-RNA heteroduplex structure formation that turns the gold colloid's ruby red color to blue due to the nano-aggregation in an acidic environment, which can be detected by the naked eye or measuring the absorbance. The DNA probe was designed to bind to a genomic RNA conserved region recognized in all four dengue serotypes. Dengue virus serotype 1 was utilized as a framework for virus detection; the designed nanosensor exhibited great specificity and selectivity, with the detection limit of ∼1 pg μL-1 (∼1.66 × 106 RNA copies per reaction) and time of analysis of about 1 h including the RNA extraction step. The proposed colorimetric nanosensor offers an alternative tool for specific and highly sensitive detection of dengue that eliminates the requirement for thermal cycling and primer sets in PCR-based assays.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

Protocol of Detection of West Nile Virus in Clinical Samples.

West Nile virus (WNV) is one of the leading causes of arboviral encephalitis in the United States but is often underdiagnosed. Despite the wide breadth of WNV-induced clinical disease syndromes, many of the symptoms associated with WNV are nonspecific at the time of presentation; thus, choosing the right diagnostic tool is essential to not only understand the true burden of disease but also provide pathogen-directed interventions for WNV-infected patients. In this chapter, we briefly discuss the three most common types of diagnostic methods for WNV in human clinical samples: nucleic acid detection, enzyme-linked immunoassay (ELISA), and plaque reduction neutralization test (PRNT) and present the method for PRNT.

Detection of respiratory viruses in clinical samples

Respiratory viral infections accounts for more than 4.25 million fatalities each year, making them the third most common cause of death worldwide. Although the majority of acute respiratory infections are assumed to be caused by viruses, the exact cause is often unknown. Comprehensive mapping of viral genomic sequencing is done, which also shows a significant degree of viral heterogeneity that contributes to the early diagnosis of respiratory illness. The development of next-generation sequencing (NGS), particularly for the detection of unidentified respiratory viruses, has revolutionized the field of novel viral genome detection.This review focusses on different models of sequencing techniques available for novel viral genome detection. Although there are still major technical and ethical issues in using this technologies for clinical detection, this technology has great promise for the future as a way to better understand respiratory viruses and make diagnoses that are more precise.

Characterization of PRRSV in clinical samples and the corresponding cell culture isolates.

Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV-2, 26 PRRSV-1, and three PRRSV-1 and PRRSV-2 PCR-positive) and their isolates in MARC-145 and/or ZMAC cells. For three PRRSV-1 and PRRSV-2 PCR-positive clinical samples, both PRRSV-1 and PRRSV-2 were isolated in ZMAC cells, whereas either PRRSV-1 or PRRSV-2, but not both, was isolated in MARC-145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty-six PRRSV-1 and most of 995 PRRSV-2 PCR-positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV-2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC-145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC-145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild-type virus strain found on the farm and instead contains vaccine-like virus. Vaccine-specific PCR and next-generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC-145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV-2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus.

Simple SummaryPorcine epidemic diarrhea virus infection is an important acute diarrheal disease of swine especially in infected piglets can caused severe diarrhea, dehydration with difficulty in digesting milk curd, leading to death. The diagnosis of this viral infection is essential for monitoring and managing the disease. There is surprisingly little evidence such as easy rapid detection in the field. In this study, we developed rapid the reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow for Porcine epidemic diarrhea virus detection targeted the membrane gene in the genome sequence of the virus. Herein, the results shown that the established assay is simple and rapid, increases high sensitivity and specificity, and can be applied in the field.Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Оценка эффективности ПЦР тест-систем для контроля деконтаминации объектов окружающей среды, потенциально инфицированных коронавирусом SARS-CoV-2

In the context of the coronavirus pandemic, one of the priority tasks is the disinfection of environmental objects in order to comply with the anti-epidemic regime. In this regard, questions arise regarding the laboratory control of the effectiveness of disinfection measures. The use of virological approaches is hardly acceptable for solving this problem, and the use of fast and affordable methods of molecular genetic analysis comes to the fore, which are certainly of signal value, but sufficient to evaluate the effectiveness of these measures. In this work, studies were carried out on the detection of SARS-CoV-2 coronavirus RNA by several test systems using the real-time polymerase chain reaction method in swabs from various surfaces after they were treated with a disinfectant. The study showed that to control the decontamination of environmental objects potentially infected with the SARS-CoV-2 coronavirus, test systems can be used to detect the RNA of the SARS-CoV-2 virus in clinical samples. The greatest sensitivity is shown for PCR kits in which multichannel detection of individual RNA sites is carried out. Keywords: disinfectants, decontamination control, novel coronavirus infection COVID-19.

Nervous necrosis virus titration and antigen quantitation by indirect sandwich enzyme linked immunosorbent assay

Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterised. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimise an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.