Gene-specific Probes Research Articles

Molecular epidemiology of avian influenza viruses and avian coronaviruses in environmental samples from migratory bird inhabitants in Bangladesh.

Migratory birds are a natural reservoir for major respiratory viruses such as the avian influenza virus (AIV) and the avian coronavirus (AvCoV). Transmission of these viruses from migratory birds to domestic birds increases the prevalence of those diseases that cause severe economic and public health concerns in Bangladesh. The study focused on active surveillance of major respiratory viral pathogens in migratory birds, molecular identification of the viruses, and their phylogenetic origin. To conduct this study, 850 environmental samples (830 fecal samples, 10 soil samples, and 10 water samples) were collected during three consecutive winter seasons from three divisions (Dhaka, Sylhet, and Mymensingh) and pooled according to the year of collection and locations, resulting in a total of 184 tested samples. Using gene-specific primers and probes in TaqMan-and SYBR Green-based RT-qPCR assays, the samples were screened for AIV and AvCoV, respectively. Out of the 184 pooled samples, 37 were found to be positive for these respiratory pathogens. Furthermore, out of the 37 (20.11%) positive respiratory pathogens, 11.96% were AIV (n = 22) and 8.15% were AvCoV (n = 15). For the first time in Bangladesh, AIV H4N2, H4N6, and AvCoVs have been found in fecal samples from migratory birds through surveillance. Phylogenetic analyses of the HA and NA genes of AIV and the polymerase gene (Orf 1) of AvCoV revealed that these strains share a close phylogenetic relationship with the isolates from wild birds in Europe and Asia. The Bangladeshi strains with Eurasian ancestry might pose a significant threat to migratory birds flying through the Asian flyways. They might also be a potential source of virus introduction and spread to poultry raised on land. These findings emphasize the significance of ongoing AIV and AvCoV surveillance in migratory birds in Bangladesh.

The relationship between neurotrophins and cognitive functions in the context of emotional response to sleep deprivation

IntroductionStudies conducted up to date on the subject of deprivation of sleep (DS) primarily focused either on its impact on certain cognitive abilities or mood-enhancing effects in patients with depression. A notable body of evidence suggests that both might be related to alterations in neurotrophin synthesis induced by DS. However, the role of NTs as an interface between DS, mood, and cognitive functions is unclear.ObjectivesThe study aimed to investigate associations between cognitive abilities measured by Trail Making Test (TMT) and Stroop Color and Wort Test (ST), serum protein concentrations of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), neurotrophin-4 (NT4) as well as the expression of their respective genes after a night of sleep deprivation.MethodsEach participant (n=76) underwent a 24-hour DS under the control of actigraphy. Venous blood collection, TMT, and ST were carried out in the morning after DS. Mood was evaluated twice, after DS and in the preceding evening; based on the alleviation of depression symptoms participants were divided into respondents (RE; n=47) and non-respondents (NR; n=29). Serum protein concentration was determined using ELISA kits. Gene expression was evaluated by quantitative real-time polymerase chain reaction with gene-specific probes (reference gene: β-actin). Relative expression was calculated using the Livak formula. TMT is a neuropsychological instrument; Part 1 is thought to evaluate mostly attention, whereas Part 2 executive functions. ST is a 2-part test applied in the assessment of response inhibition and complex attention.ResultsIn RE, cognitive abilities were not associated with expression levels of any of the studied proteins or mRNA (all p>0.05). In NR, BDNF and GDNF mRNA expressions negatively correlated with TMT Part 1 (p=0.017, p=0.048, respectively); scores obtained in TMT Part 2 bore a similar relation to BDNF, GDNF, and NT4 mRNA (p=0.034, p=0.041, p=0.026, respectively). In this group, expression of all BDNF, GDNF, NT3, NT4 mRNA correlated negatively with both parts of ST (p<0.001, p=0.009, p=0.042, p=0.009 for Part 1; p<0.001, p=0.003, p=0.031, p=0.014 for Part 2, respectively).ConclusionsThose results suggest that alterations in the synthesis of NTs might be an element of the molecular milieu characterizing different types of DS response. Negative correlations between test scores and NT mRNA expressions could imply that the reduction of the production of NT proforms might protect against the decline of cognitive functions in the aftermath of DS. Projects using a larger battery of tests as well as analyzing immature forms of NTs would be desirable in order to further investigate mechanisms underlying DS response.Disclosure of InterestNone Declared

Abstract B032: Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions

Abstract Chromosomal instability (CIN) is the most common form of genomic instability in cancer. CIN leads to structural and numerical chromosomal aberrations and to heterogeneity of such aberrations inside the same tumor. Together with other forms of genomic and epigenomic instability, CIN underlies the most dangerous feature of cancer cells, namely, ability to evolve, allowing cancer cells to evade immunologic surveillance, to metastasize, to become resistant to drug treatments. Studies of chromosomal instability are significantly impeded by the lack of comprehensive approaches for simultaneous detection of different forms of ongoing chromosomal instability and their outcomes. We developed an approach that allows to analyze the clonal composition and chromosomal heterogeneity in cell lines and cancer samples, as well as rates of different forms of ongoing chromosomal instability in cancer cell lines. At the heart our analysis is a multiplex interphase FISH (miFISH) with seven sequential hybridizations of color probe panels resulting in the assessment of a total of 35 probes per single cell. Each probe panel is designed to include the centromeric probe and probes on the p- and q-arm for a given chromosome that allows for the simultaneous assessment of centromeric probes and gene-specific arm probes. This approach was applied to interphase nuclei, metaphase spreads and bi-nucleated/dividing cancer cells in colorectal and cervical cell lines and cancer samples. miFISH allows for automated acquisition of nuclei and metaphase spreads on a BioView spotcounting system. A gallery provides detailed views for semi-automatic as well as manual analysis of the obtained images. A total of 10000-25000 cell images were collected from each slide. The simultaneous evaluation of our strategically chosen centromeric and gene-specific probes within the same cells allows unprecedented insights in the patterns of distribution of genetic material among cancer cells in cancer cell population, and, especially, in dividing cells. We evaluated clonality, clonal and non-clonal chromosomal heterogeneity, ploidy distributions, and occurrence of endoreplication. Assessment of dividing cells based on equal or unequal distribution of miFISH centromeric and gene-specific signals allowed to estimate and compare the rates and outcomes of different forms of ongoing chromosomal instability such as structural rearrangements and amplifications, loss of chromosomes or chromosomal fragments in micronuclei, non-disjunctions, grossly asymmetrical cell divisions (including multipolar mitoses). Conclusions: Gradual changes in chromosomal/genomic content of cancer cells occur due to structural rearrangements, losses of chromosomes and chromosomal fragments in micronuclei, and by relatively rare single chromosome non-disjunctions. Grossly abnormal cell divisions (massive non-disjunctions and structural rearrangements) is the main source for the high level of chromosomal abnormalities and aneuploidy in evolved cancer cell populations. Citation Format: Anna Roschke. Comprehensive analysis of clonality, segregation errors, chromosomal instability and heterogeneity in cancer cell lines and cancer samples reveals that cancer evolution is often promoted by highly aberrant cellular divisions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B032.

A TiO2-adenine nanocomposite as modification material for screen-printed gold electrode to detect H1N1 (swine flu) virus: A disposable genosensor

A genosensor based on TiO2-adenine nanocomposites (NCs) modified screen-printed gold electrode surface (TA-SPGE) was immobilized with haemagglutinin (HA) gene specific probe containing 5′ amine group and investigated for swine flu virus (H1N1). Bare SPGE surface was modified by drop coating of the nanocomposites. The NCs and precursors were characterized structurally and morphologically by adopting various techniques such as Fourier Transform Infrared (FTIR) spectra, X-ray Diffraction (XRD) spectra, UV–Visible spectra, Raman spectra, Zeta sizer, Field-Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive spectra (EDS) and High-Resolution Transmission Electron Microscopy (HR-TEM) with Selected Area Electron Diffraction (SAED) pattern. Electrochemical techniques termed as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) were employed for analytical detection of single-stranded viral genome in phosphate buffer saline (pH-7.2) by using methylene blue (MB) redox indicator. Moreover, the performance of designed electrode was optimized at different concentration, pH, and scan rates. The proposed genosensor revealed high sensitivity 10.6 μA/ng.cm2, lower detection limit (LOD) of 6.7 × 10-8 ng/mL and wide linear range of 3 × 10-8-3 × 10-3 ng/mL with regression coefficient of R2 = 0.989 towards swine flu virus DNA. Additionally, the modified genosensor revealed good stability and specificity against human DNA and various pathogens such as H3N2 influenza virus, Bacillus subtilis, Neisseria meningitidis, Staphylococcus aureus and Streptococcus pyogenes. The proposed genosensor is based on relatively simple methodology, quite amicable and could promptly be used as point-of-care test for swine flu virus detection.

A-271 Verification of a multiplexed STI Pathogen Microarray Test with Saliva Collected With SDNA-1000 Kit

Abstract Background Sexually transmitted infections (STI) spread through oral sex potentially cause infections of the mouth, throat, genitals, or rectum. Saliva may be an important source of transmission of STI following oral sex. Multiplexed STI assays are useful diagnostically for patients with risk factors for STI as co-infections occur frequently. Methods Alimetrix STI is a multiplexed laboratory developed test for the identification of STI pathogens present in clinical specimens. DNA was collected in SDNA-1000 devices, extracted, purified, underwent multiplex PCR, and labeled amplicons were hybridized to a microarray with gene-specific 50mer capture probes. Fluorescently tagged amplicons were detected on a microarray scanner. Verification included analytical sensitivity (Limit of Detection (LOD), competition, and inclusivity), stability, and analytical specificity (exclusivity, precision and accuracy). Pathogens included in the multiplexed microarray included N. gonorrhea, C. trachomatis, T. vaginalis, T. pallidum , herpes 1 and 2, M. genitalium, U. urealyticum, and monkeypox. Results LOD for each pathogen in the Spectrum Solutions SDNA Collection System, throat swab, and first catch urine is found in Table 1. Competition of organisms showed the ability to co-detect up to 5 organisms from a single sample. Saliva collected using Spectrum Solutions SDNA Collection System was stable for 35 days at room temperature enabling discreet at home collection. Inclusivity showed related strains were detectable at 1× LOD, while maintaining 100% precision, 100% accuracy, and 97% exclusivity for high concentrations of closely related analytes in normal flora. Conclusion Control of STI associated with oral sex is dependent upon the demonstration of all pathogens present. Saliva collected in the SDNA-1000 device a high degree of sensitivity, precision, and accuracy, as well as the ability to detect multiple analytes (co-infection) within a given sample. Saliva is a clinically informative specimen source useful for the diagnosis, monitoring, and management of patients with oral and systemic disease.

TiO2-guanine as a new amalgamation compoundfor fabrication of a disposable biosensor with high sensitivity and rapid detection of H1N1 swine flu virus.

A TiO2-guanine nanocomposite (TG NC)-based electrochemical biosensor was immobilized with hemagglutinin (HA) gene specific probe with 5' NH2 group on screen-printed gold electrode (probe(ss)DNA-TG-SPGE). The modified biosensor was examined for H1N1 swine flu virus. TG NCs along with precursors were characterized spectroscopically and morphologically by employing several approaches. Electrochemical investigations were performed with the help of cyclic voltammetric (CV) and electrochemical impedance spectroscopy (EIS) in 0.1 M phosphate buffer saline (PBS; pH 7.4) with 1 µM methylene blue (MB) redox indicator. For better detection of single-stranded virus DNA, the modified electrode was optimized at various concentrations, pH, and scan rates. The modified biosensor showed high sensitivity (40.32 μA/ng.cm2), low LOD (0.00024 ng/6 µL), and broad linear range 0.0002-20 ng/6µL with coefficient of determination ofR2=0.9981 for H1N1 virus detection. The HA gene-modified biosensor presented decent stability and specificity against different infectious pathogens including H3N2 virus and human DNA with negative response. Furthermore, the modified biosensor also responded well for real sample target DNA detection with a recovery of >96%. The simply designed HA gene-modified biosensor transduces decreased current response towards target-specific (ss)DNA binding and could be used as a rapid detection tool for H1N1 swine flu virus diagnosis.

Carbohydrate-coated magnetic and gold nanoparticles for point-of-use food contamination testing

With a significant part of the world facing multiple foodborne outbreaks each year, rapid and affordable detection technologies are needed. This paper presents simple equipment-free isolation of bacteria from foods using glycan-coated Magnetic Nanoparticles (gMNP) and their genomic detection using dextrin-capped Gold Nanoparticles (GNPs) in <7h. The synthesized gMNP and GNPs can be stored at room temperature and do not require expensive recognition biomolecules for bacterial identification. Isolation of E. coli directly from leafy green vegetables was achieved in <60 min, and its binding to gMNP was confirmed using Transmission Electron Microscopy (TEM). As-synthesized GNPs were found to be <50 nm in diameter and showed a characteristic wavelength peak at 520 nm in the visible spectrum. A PCR-less GNP biosensor was developed targeting the uidA gene allowing rapid bacterial detection in <30 min and successfully differentiating (p<0.05) E. coli from S. Enteritidis, K. pneumoniae, and E. cloacae. Hybridization between the uidA gene-specific probe and target DNA resulted in the stability of GNPs under an acidic environment, while their aggregation in the absence of target DNA was observed. Successful detection was achieved at the initial bacterial contamination as low as 5.2 X 102 and 6.3 X 102 CFU/mL from artificially contaminated spinach and lettuce samples, respectively.

Inferring spatial transcriptomics markers from whole slide images to characterize metastasis-related spatial heterogeneity of colorectal tumors: A pilot study

Over 150 000 Americans are diagnosed with colorectal cancer (CRC) every year, and annually over 50 000 individuals will die from CRC, necessitating improvements in screening, prognostication, disease management, and therapeutic options. Tumor metastasis is the primary factor related to the risk of recurrence and mortality. Yet, screening for nodal and distant metastasis is costly, and invasive and incomplete resection may hamper adequate assessment. Signatures of the tumor-immune microenvironment (TIME) at the primary site can provide valuable insights into the aggressiveness of the tumor and the effectiveness of various treatment options. Spatially resolved transcriptomics technologies offer an unprecedented characterization of TIME through high multiplexing, yet their scope is constrained by cost. Meanwhile, it has long been suspected that histological, cytological, and macroarchitectural tissue characteristics correlate well with molecular information (e.g., gene expression). Thus, a method for predicting transcriptomics data through inference of RNA patterns from whole slide images (WSI) is a key step in studying metastasis at scale. In this work, we collected tissue from 4 stage-III (pT3) matched colorectal cancer patients for spatial transcriptomics profiling. The Visium spatial transcriptomics (ST) assay was used to measure transcript abundance for 17 943 genes at up to 5000 55-micron (i.e., 1–10 cells) spots per patient sampled in a honeycomb pattern, co-registered with hematoxylin and eosin (H&E) stained WSI. The Visium ST assay can measure expression at these spots through tissue permeabilization of mRNAs, which are captured through spatially (i.e., x–y positional coordinates) barcoded, gene specific oligo probes. WSI subimages were extracted around each co-registered Visium spot and were used to predict the expression at these spots using machine learning models. We prototyped and compared several convolutional, transformer, and graph convolutional neural networks to predict spatial RNA patterns at the Visium spots under the hypothesis that the transformer- and graph-based approaches better capture relevant spatial tissue architecture. We further analyzed the model’s ability to recapitulate spatial autocorrelation statistics using SPARK and SpatialDE. Overall, the results indicate that the transformer- and graph-based approaches were unable to outperform the convolutional neural network architecture, though they exhibited optimal performance for relevant disease-associated genes. Initial findings suggest that different neural networks that operate on different scales are relevant for capturing distinct disease pathways (e.g., epithelial to mesenchymal transition). We add further evidence that deep learning models can accurately predict gene expression in whole slide images and comment on understudied factors which may increase its external applicability (e.g., tissue context). Our preliminary work will motivate further investigation of inference for molecular patterns from whole slide images as metastasis predictors and in other applications.

Digital PCR: A Tool to Authenticate Herbal Products and Spices.

Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.

Development of amperometric biosensor based on cloned hemagglutinin gene of H1N1 (swine flu) virus.

The recent emergence of respiratory viruses especially COVID-19 and swine flu has underscored the need for robust and bedside detection methods. Swine flu virus is a very infectious virus of the respiratory system. Timely detection of this virus with high specificity and sensitivity is crucial for reducing morbidity as well as mortality. Cloning of gene segments into a non-infectious agent helps in the development of detection methods, vaccine development, and other studies. In this study, cloning was used to develop a biosensor for H1N1 pdm09 detection. A segment of the hemaglutinin gene was cloned in a vector and characterized with the help of colony touch PCR and blue–white screening. The recombinant plasmid was extracted, and the gene segment was confirmed with the help of HA-specific primers. A 5′ amine group-attached hemagglutinin (HA) gene-specific DNA probe was immobilized on the working gold electrode surface to make a quick, specific, reliable, and sensitive detection method for H1N1pdm09 virus in human nasal swab samples. The HA probe was immobilized on the cysteine applied gold electrode of the screen-printed electrode through 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Differential pulse voltammetry was performed with the help of methylene blue, which is a redox indicator for the detection of single-stranded cloned HA gene segment. The developed sensor depicted high sensitivity for the H1N1 influenza virus with a detection limit of 0.6 ng ssDNA/6 µl of the cloned HA sample. Specificity was also checked using H3N2 virus, N. meningitides, influenza A and positive H1N1pdm09 samples.

Prevalence of Newcastle Disease Virus in Wild and Migratory Birds in Haryana, India.

Newcastle disease virus (NDV) can infect approximately 250 avian species and causes highly contagious Newcastle disease (ND) in domestic poultry, leading to huge economic losses. There are three different pathotypes of NDV, i.e., lentogenic, mesogenic, and velogenic. Wild resident (wild) and migratory birds are natural reservoirs of NDV and are believed to play a key role in transmitting the virus to domestic poultry. The present study was conducted to determine the prevalence of NDV in wild and migratory birds in the state of Haryana, India, during two migratory seasons (2018-19 and 2019-20). In total 1379 samples (1368 choanal swabs and 11 tissue samples) were collected from live (n = 1368) or dead birds (n = 4) belonging to 53 different avian species. These samples belonged to apparently healthy (n = 1338), sick (n = 30), and dead (n = 4) birds. All samples were tested for NDV by real-time reverse transcription-PCR using M gene specific primers and probe. Of the 1379 samples, 23 samples from wild birds [Columba livia domestica (n = 12, 52.17%), Pavo cristatus (n = 9, 39.13%), and Psittaciformes (n = 2, 8.69%)] were found positive for NDV. Only one of the 23 samples (from P. cristatus) was positive for F gene, indicating it to be a mesogenic/velogenic strain. These results indicate that both lentogenic and velogenic strains of NDV are circulating in wild birds in Haryana and that further studies are needed to characterize NDV strains from wild/migratory birds and domestic poultry to determine the extent of virus transmission among these populations. This study considers the disease transmission risk from domestic pigeons and parrots to commercial poultry and vice versa, and the results emphasize the need for strict biosecurity strategies to protect commercial poultry in the region.

Cover: Journal of Basic Microbiology. 3‐4/2022

Journal of Basic MicrobiologyVolume 62, Issue 3-4 p. 197-197 COVER PICTUREFree Access Cover: Journal of Basic Microbiology. 3-4/2022 First published: 21 March 2022 https://doi.org/10.1002/jobm.202270006AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Cover illustration: The cover picture shows nitrite oxidizers and ammonium oxidizers. These types of bacteria serve to reduce nitrogen compounds in wastewater. The vital bacterial cells were marked with specific gene probes and then photographed using fluorescence microscopy. (Image: Helga Gehrke, Kooperationslabor von Ruhrverband, Emschergenossenschaft und Lippeverband (laboratory run by the Ruhrverband in cooperation with the Emscher Cooperative and the Lippe Association), Essen, Germany) Volume62, Issue3-4Special Issue: Emerging microbial technologies for wastewater treatmentMarch-April 2022Pages 197-197 RelatedInformation

Accuracy and applications of sequencing and genotyping approaches for CYP2A6 and homologous genes.

We evaluated multiple genotyping/sequencing approaches in a homologous region of chromosome 19, and investigated associations of two common 3'-UTR CYP2A6 variants with activity in vivo. Individuals (n = 1704) of European and African ancestry were phenotyped for the nicotine metabolite ratio (NMR), an index of CYP2A6 activity, and genotyped/sequenced using deep amplicon exon sequencing, SNP array, genotype imputation and targeted capture sequencing. Amplicon exon sequencing was the gold standard to which other methods were compared within-individual for CYP2A6, CYP2A7, CYP2A13, and CYP2B6 exons to identify highly discordant positions. Linear regression models evaluated the association of CYP2A6*1B and rs8192733 genotypes (coded additively) with logNMR. All approaches were ≤2.6% discordant with the gold standard; discordant calls were concentrated at few positions. Fifteen positions were discordant in >10% of individuals, with 12 appearing in regions of high identity between homologous genes (e.g. CYP2A6 and CYP2A7). For six, allele frequencies in our study and online databases were discrepant, suggesting errors in online sources. In the European-ancestry group (n = 935), CYP2A6*1B and rs8192733 were associated with logNMR (P < 0.001). A combined model found main effects of both variants on increasing logNMR. Similar trends were found in those of African ancestry (n = 506). Multiple genotyping/sequencing approaches used in this chromosome 19 region contain genotyping/sequencing errors, as do online databases. Gene-specific primers and SNP array probes must consider gene homology; short-read sequencing of related genes in a single reaction should be avoided. Using improved sequencing approaches, we characterized two gain-of-function 3'-UTR variants, including the relatively understudied rs8192733.

Development of transgenic cucumber mosaic virus (CMV) resistant gerbera plants expressing CMV coat protein gene

Gerbera (Gerbera jamesonii L.) has its immense importance to the floriculture industry worldwide. The gerbera flower production has been hampered by various viruses, among them cucumber mosaic virus (CMV) has shown considerable damage.As natural resistance to CMV is absent in gerbera, here, we have made an attempt to develop transgenic gerbera plants expressing coat protein (CP) gene of CMV via Agrobacterium mediated transformation of base petiole explants for genetic resistance to CMV infection. Among the 44 putative transgenic gerbera plant acclimatized, 39 were found positive for integration of CP gene by polymerase chain reaction and southern hybridization assay using their specific primer and probe respectively. Northern hybridization assay using CP gene specific probe confirmed the transcription of transgene in all 39 transgenic plants. These plants showed translation of CP during DAS-ELISA when tested with antiserum specific to CP of CMV. These 39 plants when challenged by mechanical inoculations with CMV gerbera isolate showed virus resistance in 53% (21 out of 39) plants, virus tolerance (delayed mild symptom) in 33% (13/39) plants, while rest 12.8% (5/39) plants showed severe disease symptoms. The CP mediated resistance of CMV in transgenic gerbera is being reported for the first time from India.

NanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro

BackgroundNext Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software.Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency. MethodsmCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM. ResultsWe found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue. ConclusionsFor viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.

Comparative analysis of point-of-care, high-throughput and laboratory-developed SARS-CoV-2 nucleic acid amplification tests (NATs)

Multiple nucleic acid amplification tests (NATs) are available for the detection of SARS-CoV-2 in clinical specimens, including Laboratory Developed Tests (LDT), commercial high-throughput assays and point-of-care tests. Some assays were just recently released and there is limited data on their clinical performance. We compared the Xpert® Xpress SARS-CoV-2 (Cepheid) and Vivalytic VRI Panel (Schnelltest COVID-19) (Bosch) point-of-care tests with four high-throughput assays and one LDT, the cobas® SARS-CoV-2 test (Roche), the Allplex™ 2019-nCoV Assay (Seegene), the SARS-CoV-2 AMP (Abbott) Kit, the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona) as well as an assay using a SARS-CoV-2 RdRP gene specific primer and probe set. Samples from patients with confirmed SARS-CoV-2 infection, samples from the first and second SARS-CoV-2-PCR External Quality Assessment (EQA) (INSTAND e.V.) and a 10-fold serial dilution of a SARS-CoV-2 cell culture (SARS-CoV-2 Frankfurt 1) supernatant were examined. We determined that the NAT assays examined had a high specificity. Assays using the N gene as target demonstrated the highest sensitivity in the serial dilution panel, while all examined NAT assays showed a comparable sensitivity when testing clinical and EQA samples.

Genosensor for rapid, sensitive, specific point-of-care detection of H1N1 influenza (swine flu)

A 5′ amine group-linked haemagglutinin (HA) gene-specific probe was attached over the surface of a working electrode to develop a rapid, specific, and sensitive point of care detection assay for H1N1 (swine flu) in human respiratory nasal swabs. The probe was attached with a cysteine covered screen-printed gold electrode via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). The electrochemical assay was performed using differential pulse voltammetry with the use of the redox indicator methylene blue for the detection of different concentrations of the single-stranded viral genome. The developed genosensor showed high sensitivity for H1N1 influenza virus with a detection limit of 0.002 ng/6 μL of viral nucleic acid in the sample. Samples were analysed by quantitative real-time Polymerase Chain Reaction as well as by conventional PCR. The genosensor showed high specificity, as no cross-reaction was observed with the heterologous nucleic acid of different pathogens (Salmonella typhi, Neisseria meningitides, and Streptococcus pyogenes) and human DNA, and it was specific for H1N1 with a sensitivity of ∼49 μA cm−2 ng-1. Genosensor is based on a very simple methodology that can be followed based on its easy-to-access approach. It is quick and could be used as a point-of-care test for the detection of influenza virus within 30 min.

Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).

Development of a polymerase chain reaction - Nucleic acid sensor assay for the rapid detection of chicken adulteration

A simple, accurate, and affordable rapid identification technology for authenticating meat species is urgently needed in the food industry. In this study, a chicken-specific polymerase chain reaction combined with a nucleic acid sensor test (Chicken-PCR-Sensor) was developed for fast identification of chicken meat, and its feasibility was evaluated. In this work, gene specific primers and probes were uniquely designed based on certain principles. Chicken mitochondrial DNA underwent targeted amplification by PCR, and the PCR product was directly hybridized to two probes in the PCR solution without need for a secondary hybridization step. The results were assessed using a nucleic acid sensor within 2–3 min. This nucleic acid sensor test provides a visual read-out format for the rapid detection of nucleic acids in field testing. The Chicken-PCR-Sensor test was able to specifically detect chicken and was able to detect as little as 0.01% adulterated chicken in the meat mixture. The robustness of the test was successfully evaluated with commercial food samples. As a promising novel molecular detection tool, this test might have potential applications for evaluating chicken adulteration and traceability given its rapidity, high sensitivity, and simple work flow.

Isolation, nucleotide sequencing and genomic comparison of a Novel SXT/R391 ICE mobile genetic element isolated from a municipal wastewater environment

Integrative Conjugative Elements (ICE’s) of the SXT/R391 family have largely been detected in clinical or environmental isolates of Gammaproteobacteria, particularly Vibrio and Proteus species. As wastewater treatment plants accumulate a large and diverse number of such species, we examined raw water samples taken from a municipal wastewater treatment plant initially using SXT/R391 family integrase gene-specific PCR probes to detect the presence of such elements in a directed approach. A positive amplification occurred over a full year period and a subsequent Restriction Fragment Length Polymorphism (RFLP) analysis revealed a very limited diversity in the treatment plant examined. Samples demonstrating positive amplification were cultured using Vibrio and Proteus selective media and PCR amplification tracking was utilized to monitor SXT/R391-ICE family containing strains. This screening procedure resulted in the isolation and identification of a Proteus mirabilis strain harbouring an ICE. Whole-genome sequencing of this ICE containing strain using Illumina sequencing technology revealed a novel 81 kb element that contained 75 open reading frames on annotation but contained no antibiotic or metal resistance determinants. Comparative genomics revealed the element contained a conserved ICE core with one of the insertions containing a novel bacteriophage defence mechanism. This directed isolation suggests that ICE elements are present in the environment without apparent selective pressure but may contain adaptive functions allowing survival in particular environments such as municipal wastewater which are reservoirs for large bacterial phage populations.