Detection Of Viral Nucleic Acids Research Articles

Rapid Detection of Virus Nucleic Acid via Isothermal Amplification on Plasmonic Enhanced Digitizing Biosensor.

Rapid detection for infectious diseases is highly demanded in diagnosis and infection prevention. In this work, we introduced a plasmonic enhanced digitizing biosensor for the rapid detection of nucleic acids. The sensor successfully achieved the detection of loop-mediated isothermal amplification for the hepatitis virus in this work. The sensor comprised a nanodisc array and Bst polymerases conjugated on the rough surface of a nanodisc. The rough surface of the nanodisc provided plasmonic hot spots to enhance the fluorescence signal. The virus DNA was detected by conducting a modified loop-mediated isothermal amplification with fluorescence resonance energy transfer reporter conjugated primers on the sensor. The modified isothermal amplification improved the signal contrast and detection time compared to the original assay. By integrating the modified amplification assay and plasmonic enhancement sensor, we achieved rapid detection of the hepatitis virus. Nucleic acid with a concentration of 10−3 to 10−4 mg/mL was detected within a few minutes by our design. Our digitizing plasmonic nanoarray biosensor also showed 20–30 min earlier detection compared to conventional loop-mediated isothermal amplification sensors.

Effectiveness of Disinfectants Suitable for Inactivating SARS-CoV-2 at Cold-Chain Temperature.

To prevent the spread of SARS-CoV-2 in cold-chain transportation in China, we developed specific cryogenic disinfectants. Carrier tests were performed against SARS-CoV-2 at − 20 °C for the four cryogenic disinfectants developed and qRT-PCR was used to test the virus RNA. Peracetic acid, chlorine disinfectants (two different concentrations), and quaternary ammonium disinfectant with their antifreeze can all inactivate SARS-CoV-2 in 5 min at − 20 °C. However, after 2–3 h of exposure, only chlorine disinfectant could destroy SARS-CoV-2 RNA. The viruses treated with peracetic acid and quaternary disinfectants showed positive Ct values even after 3 h detected with qRT-PCR. The conclusion was that the cold-chain disinfectants we tested could inactivate SARS-CoV-2 quickly and effectively, but only chlorine disinfectants could destroy nucleic acids in 3 h. Our study also illustrated that using qRT-PCR detection of viral nucleic acids to assess disinfection was inappropriate.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12560-022-09509-0.

Paper-based detection of Epstein-Barr virus using asymmetric polymerase chain reaction and gold silicon particles

A new paper-based lateral flow nucleic acid (LFNA) test platform was established in this study using asymmetric polymerase chain ceaction (A-PCR) for signal amplification. This new method allowed a visual detection of Epstein-Barr virus (EBV) nucleic acids with high specificity and low cost. In addition, as part of our strategy we employed a sandwich system of capture probe (CP)/gold nanoparticles (AuNPS) and silicon dioxide (SiO2) (AuNPS@SiO2) nanospheres/target DNA/avidin complexes as the sensing platform. Biotin-labeled target DNA was obtained by A-PCR and later introduced in the LF device. The CP/target DNA/AuNPS@SiO2 complexes were captured on the test zone by the specific reaction between biotin and avidin, and the remaining CP/AuNPS@SiO2 particles were captured on the quality control zone by the hybridization between CP and a quality control probe. Au@SiO2 accumulation in the test and quality control zones of the device enabled a visual detection of the specific target sequences. The method detection limit was 50 nM of the target DNA, which was lower than that of the LFNA biosensor (LFNAB) without PCR amplification and Au@SiO2 particles. In conclusion, the novel paper-based platform described here is a low cost, efficient and fast visual detection method that offers high sensitivity and other benefits compared to alternative methods in use.

Magnetic-Responsive Surface-Enhanced Raman Scattering Platform with Tunable Hot Spot for Ultrasensitive Virus Nucleic Acid Detection.

Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.

Modern Advances on the Diagnosis of Bovine Viral Diarrhoea Virus In Camelids

Bovine viral diarrhoea virus (BVDV) is one of the most leading causes of diarrhoea in many species of animals including camelids. Special attention is being paid to studying various aspects of the BVDV infection, pathogenesis, epidemiology, diagnosis and vaccines in the bovine species. Little is still known about these aspects in camelids especially dromedaries. The development of accurate diagnostic tests is a critical step for the identifcation of infected animals, especially persistently infected and for the control and eradication of BVDV. Although adaptation of the currently used diagnostic assays for the virus in the bovine and ovine species for the diagnosis of BVDV in the camelids, it is highly recommended to develop novel specifc diagnostic assays of the virus in camels. There are several strategies for the diagnosis of BVDV including virus isolation, detection of viral (antigens, antibodies and nucleic acids) and identifcation of the circulating strains of the virus by doing sequencing and phylogenetic analysis. Identifcation of the persistently infected animals is one of the main challenges facing the diagnosis and control of the virus. Our main objectives are to highlight the most recent advances in the feld of BVDV diagnosis, currently used diagnostic assays and suggested some specifc approaches for the diagnosis of BVDV in camelids. Fine-tuning the currently used diagnostic assays and development of other novel specifc assays to detect BVDV infection in dromedary camels will have a substantial positive impact on the control of BVDV infections in camels.

A colorimetric sandwich-type bioassay for SARS-CoV-2 using a hACE2-based affinity peptide pair

The metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the SARS-CoV-2 receptor required for viral entry based on its specific recognition of the spike protein receptor binding domain (S_RBD) on SARS-CoV-2. We constructed a human ACE2 (hACE2)-based peptide pair by ligating discontinuous key residues involved in the hACE2–S_RBD interaction. We firstly performed in silico simulations to identify a 12-mer and 15-mer peptide pair with capability to bind to the SARS-CoV-2 S_RBD via different binding sites. Then, the bio-layer interferometry validated the specific interactions between the peptides and S_RBD, with affinities at the nanomolar level. Lastly, we developed a colorimetric sandwich-type bioassay based on S_RBD-specific peptide-modified gold nanoparticles and found the colorimetric bioassay offered fast (<30 min), simple, and sensitive detection of S_RBD protein at levels as low as 0.01 nM (0.26 ng mL-1) in SARS-CoV-2. The linear signals ranging from 105 to 107 virus copies mL-1 were achieved in typical types of environmental waters spiked with lysed SARS-CoV-2 pseudovirus. The technology can serve as a beneficial supplement to the routine virus nucleic acid detection in environment media and wastewater treatment.

Ultrasensitive biochemical sensors based on controllably grown films of high-density edge-rich multilayer WS2 islands

Preferentially exposed active edges are advantageous in the application of two-dimensional materials (2DMs) in high-performance biochemical sensors. Despite current progresses, controllable large-scale production of edge-rich 2DM films for practical applications remains a challenge. In this work, we report the controllable single-step growth of wafer-scale edge-rich WS2 films of high-density nanoscale multilayer WS2 islands and their application in sensitive, low detection limit biochemical sensors. The abundant edge active sites are shown to be crucial to their superior biochemical sensing performance. Investigations reveal that high-density edge S-active sites enable the highly sensitive attomolar-level Hg2+ detection as direct target probes via selective Hg-S bonding. Furthermore, 1 fM level selective detection of target viral nucleic acids (RdRp, ORF1ad) are achieved via functionalization of active edge sites with cDNA receptors. Our investigations provide a comprehensive method for application of similar edge-rich 2DM films in high-performance sensors for broad ranges of analytes.

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR–Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR–Cas-based nucleic acid detection strategies and the application of CRISPR–Cas as a potential antiviral agent in the context of COVID-19.

Prevention and Management of COVID -19

According to the WHO, total confirmed cases of Coronavirus up to October 07, 2021: 237,091,792 , Deaths: 4,840,927 , and total Recovered case 214,263,057 from COVID-19, triggered by the novel SARS-CoV-2 , the rate of case in major countries USA44,918,565 India33,894,312 Brazil21,517,514 Russia 7,662,560 Italy4,689,341 keeps expanding geographically. SARS-CoV-2 is a 29.9 kb RNA beta-coronavirus that is enveloped, positive-sense, and single-stranded. Close contact with infected patients and exposure to aerosol viruses from medical devices and procedures, such as endoscopy, may put healthcare workers at risk of infection. Current data suggests that children under the age of 18 have a low rate of infection (2.4 present). The current gold standard and confirming test for COVID19 is the detection of viral nucleic acid using reverse transcriptase polymerase chain reaction (RTPCR). The World Health Organization (WHO) has indicated that controlling infectious diseases like COVID19 requires education, isolation, prevention, transmission control, and treatment of sick people. By following the steps one can help to keep the illness from spreading. Protection consists of staying at home (home quarantine) and avoiding direct contact with healthy (potentially asymptomatic) or diseased patient.

Serological Diagnosis of Dengue.

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4°C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20°C or -70°C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA).

The type I interferonopathies: 10 years on.

As brutally demonstrated by the COVID-19 pandemic, an effective immune system is essential for survival. Developed over evolutionary time, viral nucleic acid detection is a central pillar in the defensive armamentarium used to combat foreign microbial invasion. To ensure cellular homeostasis, such a strategy necessitates the efficient discrimination of pathogen-derived DNA and RNA from that of the host. In 2011, it was suggested that an upregulation of type I interferon signalling might serve as a defining feature of a novel set of Mendelian inborn errors of immunity, where antiviral sensors are triggered by host nucleic acids due to a failure of self versus non-self discrimination. These rare disorders have played a surprisingly significant role in informing our understanding of innate immunity and the relevance of type I interferon signalling for human health and disease. Here we consider what we have learned in this time, and how the field may develop in the future.

Electrochemical sensors for the detection of SARS-CoV-2 virus

Coronavirus (COVID-19), a deadly pandemic has spread worldwide and created many global health issues. Though methods of its detection are being continuously developed for the early detection and monitoring of COVID-19, still there is need for more novel methods. The presently used methods include rapid antigen tests, serological surveys, reverse transcription-polymerase chain reaction (RT-PCR), artificial intelligence-based techniques, and assays based on sensors/biosensors. Of all these, RT-PCR test has high sensitivity and specificity though it requires more time for testing and need for skilled technicians. Recently, electrochemical sensors have been developed for rapid monitoring and detection of SARS-CoV-2 from the patient’s biological fluid samples. This review covers the recently developed electrochemical sensors that are focused on the detection of viral nucleic acid, immunoglobulin, antigen, and the entire viral particles. In addition, we also compare and assess their detection limits, sensitivities and specificities for the identification and monitoring of COVID-19. Furthermore, this review will address the best practices for the development of electrochemical sensors such as electrode fouling, limit of detection/limit of quantification determination and verification.

A homogeneous nucleic acid assay for simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle inductively coupled plasma mass spectrometry

In recent years, single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has become a powerful tool for biological quantitative analysis. Homogeneous analysis method requires no separation and washing steps, which is suited for the analysis of highly infectious pathogens, so as to reduce the risk of infection during the operation. SARS-CoV-2 spreads all over the world, and its early infection symptoms are similar to influenza, which brings inconvenience to triage. Therefore, developing novel analytical method for simultaneous detection of multiple viral nucleic acids is essential. Taking the advantages of SP-ICP-MS and homogeneous analysis strategy, a SP-ICP-MS homogeneous nucleic acid assay by using gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) probes was established for simultaneous sensitive analysis of SARS-CoV-2 and influenza A (H3N2). In the present of target SARS-CoV-2 or H3N2 nucleic acids, corresponding Au NPs or Ag NPs probes form larger aggregates, resulting in increased pulse signal intensity and reduced pulse signal frequency of the corresponding NPs in SP-ICP-MS measurement. In this assay, the reaction system of Au NPs and Ag NPs probes does not interfere with each other, and there was no separation and washing procedure, which facilitates operation, saves the analysis time, and improves the analysis efficiency. The linear range of this method is 5–1000 pmol L−1, with low-level limits of quantification of target nucleic acid. The developed SP-ICP-MS simultaneous homogeneous detection method has a good potential for detecting nucleic acid, protein, cell and other biological samples by changing different modification sequences on the NPs probes.

Genetic diversity of canine parvovirus variants circulating in Nigeria.

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes severe and fatal gastrointestinal disease in dogs. Lately, several mutations affecting viral protein (VP) capsid resulting in highly pathogenic variants with distinctive immunological and clinicopathological characteristics abound. This study involved screening stools of 44 randomly selected clinical cases of canine gastroenteritis from 4 cities (Ibadan, Jos, Makurdi, and Zaria) in Nigeria for CPV antigen using an on-the-spot immunoassay test kit, as well as, molecular detection of viral nucleic acid by polymerase chain reaction. Subsequently, nucleic acid sequencing of 1195-bp amplicons encompassing the VP2 encoding region was done. The resultant 40 high-quality amino acid sequences obtained were analysed for the identification and grouping of the viruses into their discrete variants - CPV-2a, CPV-2b, or CPV-2c, using key amino acids substitutions - Asn, Asp, or Glu respectively at position 426 of the VP2 gene. One-third (11/40; 27.5%) of the analysed sequences were identified as CPV-2a and two-third (29/40; 72.5%) as CPV-2c. The original CPV and CPV-2b were not detected. Also, the "new CPV-2a variant" with mutation S297A identified had two additional mutations (Y324I and T440A) associated with selective pressure and vaccination failure in their sequences. Similarly, unique CPV-2c mutants carrying genetic markers (S297A, Y324I, and Q370R) that are highly related to CPVs of Asian origin were observed. These findings revealed a high level of divergence of existing CPVs in circulation; suggesting that CPV is rapidly evolving in Nigeria lately.

Technological tools and Biosensors for detection and diagnosis of COVID-19

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a novel coronavirus. There is a constant increase in number of positive cases for COVID-19. The objective of this review is to study technology tools and sensors available for detection and diagnosis of COVID-19. COVID -19 pandamic has created surge demand for essential health care instrumentation, medicines along with technology and tools for proper control and management of corona virus. Diagnosing and screening of COVID-19 is possible using nucleic acid testing and CT scans. In this study, we have described Field-effect transistorbased graphene sensor and Plasmonic Photothermal(PPT) biosensor for highly accurate SARS-CoV-2 detection. A dual-functional localized surface plasmon resonance (LSPR) biosensor combining the photothermal effect and plasmonic sensing transduction is used for SARS-CoV-2 viral nucleic acid detection. FET based sensor for COVID- 19 used a graphene channel coupled to the SARS-CoV- 2 spike antibody with the efficient gating effect of the FET through phosphate -buffered saline (PBS; pH 7.4) buffer as the electrolyte covering. The aqueous solution-gated FET system detects SARS-CoV-2 based on electrical signal due to changes in channel surface potential. Industry 4.0 technologies such as Artificial Intelligence (AI), Internet of things(IOT) have helped in early detection and diagnosis. AI can build an intellegent platform with neural network for automatic monitoring and prediction of the spread of virus.IOT can assist in diagnosis and treatment of COVID-19 patients and can help in setting in advanced clinical work. Industry 4.0 technologies improve the working efficiency of the health care professional and provide better solution.

Current challenges in COVID-19 diagnosis: a narrative review and implications for clinical practice

Early diagnosis of coronavirus disease 2019 (COVID-19) is crucial to early treatment and quarantine measures. In this narrative review, diagnostic tools for COVID-19 diagnosis and their main critical issues were reviewed. The COVID-19 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test is considered the gold standard test for the qualitative and quantitative detection of viral nucleic acid. In contrast, tests can be used for epidemiological surveys on specific communities, including occupational cohorts, but not for clinical diagnosis as a substitute for swab tests. Computed tomography (CT) scans can be useful for the clinical diagnosis of COVID-19, especially in symptomatic cases. The imaging features of COVID-19 are diverse and depend on the stage of infection after the onset of symptoms. CT sensitivity seems to be higher in patients with positive RT-PCR. Conventional chest sensitivity shows a lower sensitivity. An important diagnostic screening tool is ultrasounds, whose specificity and sensitivity depend on disease severity, patient weight, and operator skills. Nevertheless, ultrasounds could be useful as a screening tool in combination with clinical features and molecular testing to monitor disease progression. Clinical symptoms and non-specific laboratory findings may be useful if used in combination with RT-PCR test and CT-scanning.

Fluidic handling system for PCR-based sample-to-answer detection of viral nucleic acids

Sample-to-answer detection of a viral infectious disease is becoming increasingly important to prevent pandemic outbreaks. However, only a few microfluidic chips that use nucleic acid isolation and detection have implemented all-in-one assays in a simple and effective platform. Here, we present a microfluidic platform that isolates and detects viral RNA of influenza A (H1N1) virus from clinical swab samples in a fully automated, effective, and highly sensitive manner. The device with a vertically movable top layer and a rotationally mobile bottom layer exploits surface tension in a solution. In this way, the device effectively orchestrates multiple functions, including transporting, mixing, and washing of reagents and magnetic beads without the need for on-chip valves, pumps, and microfluidic networks that increase operational and structural complexity. With integrated heating and detection functions, the device provides fully automated sample-to-answer detection with real-time polymerase chain reaction in 60 min for positive results with high sensitivity with the limit of detection of 26 copies/reaction. Our platform is useful for accurate diagnosis of infectious diseases in point-of-care settings.

REVIEW OF MEASLES VIRUS

Measles is a highly contagious, acute febrile illness that results from infection with measles virus (MV). MV is a single-stranded, negative-sense RNA virus in the genus Morbillivirus of the family Paramyxoviridae. The wild-type MV consists of 24 genotypes, three of them (B3, D8 an H1) have dominated circulation in the world. MV is transmitted by the respiratory route and illness begins with fever, cough, conjunctivitis followed by a rash and measles enanthem (Koplik spot). Laboratory confirmation of measles is provided by serological (ELISA test for detection of IgM and IgG antibodies), molecular (detection of viral nucleic acid) and viral isolation in Vero/hSLAM cells methods. As a vaccine-preventable infection, measles has a global importance and is a target of WHO strategic goals in the European region. Despite significant progress in measles control in recent years, it is necessary to improve the national vaccination coverage, and the epidemiological and laboratory monitoring of the infection. High vaccination coverage across all of the population is crucial to reach the goals of measles elimination.

Safety and efficacy of a Bluetongue inactivated vaccine (serotypes 1 and 4) in sheep

A new inactivated vaccine against Bluetongue virus (BTV) serotypes 1 and 4, was developed from field isolates. Safety and efficacy of the vaccine were evaluated in sheep by serological monitoring and virus nucleic acid detection after experimental infection of vaccinated animals. Seroconversion was observed in vaccinated animals at day 14 post vaccination (pv) with neutralizing antibody titer of 1.9 and 1.8 for serotypes 1 and 4, respectively. The titer increase significantly after the booster reaching 2.7 and persist one year >1.5 for both serotypes. After challenge with virulent isolates, vireamia was recorded in control animals, as evident by q-PCR with threshold cycles (Ct) ranging from 24 to 31 and peaked at day 10 post challenge, while no vireamia was detected in vaccinated animals. Vaccinated sheep were fully protected against the disease and infection.

Computer-aided detection of COVID-19 from CT scans using an ensemble of CNNs and KSVM classifier.

Corona Virus Disease-2019 (COVID-19) is a global pandemic which is spreading briskly across the globe. The gold standard for the diagnosis of COVID-19 is viral nucleic acid detection with real-time polymerase chain reaction (RT-PCR). However, the sensitivity of RT-PCR in the diagnosis of early-stage COVID-19 is less. Recent research works have shown that computed tomography (CT) scans of the chest are effective for the early diagnosis of COVID-19. Convolutional neural networks (CNNs) are proven successful for diagnosing various lung diseases from CT scans. CNNs are composed of multiple layers which represent a hierarchy of features at each level. CNNs require a big number of labeled instances for training from scratch. In medical imaging tasks like the detection of COVID-19 where there is a difficulty in acquiring a large number of labeled CT scans, pre-trained CNNs trained on a huge number of natural images can be employed for extracting features. Feature representation of each CNN varies and an ensemble of features generated from various pre-trained CNNs can increase the diagnosis capability significantly. In this paper, features extracted from an ensemble of 5 different CNNs (MobilenetV2, Shufflenet, Xception, Darknet53 and EfficientnetB0) in combination with kernel support vector machine is used for the diagnosis of COVID-19 from CT scans. The method was tested using a public dataset and it attained an area under the receiver operating characteristic curve of 0.963, accuracy of 0.916, kappa score of 0.8305, F-score of 0.91, sensitivity of 0.917 and positive predictive value of 0.904 in the prediction of COVID-19.