Assay For Detection Research Articles

Detection and differentiation of fowl adenovirus serotype 4 and duck adenovirus 3 using high resolution melting curve assay

Fowl adenovirus type 4 (FAdV-4) and duck adenovirus type 3 (DAdV-3) are the causative agents of clinical diseases in poultry and have caused considerable economic losses to the waterfowl industry in China. Both FAdV-4 and DAdV-3 are classified into the genus Aviadenovirus under the family Adenoviridae. The high-resolution melting (HRM) assay has become a useful method for virus genotyping, which offers the possibility of rapidly developing a differentiation technique in which the melting profile depends on the GC content of the product in the qPCR platform. The aim of this study was to develop a qPCR-HRM assay for sensitive FAdV-4 and DAdV-3 detection and differentiation. Here, specific primers were designed on the basis of the 100 K genes of FAdV-4 and DAdV-3, and a qPCR-HRM assay was established through optimization of the reaction conditions. A specificity test revealed that this method could detect only FAdV-4 and DAdV-3, with no cross-reaction with other common duck-derived viruses. A sensitivity test revealed that the lowest detection limits of FAdV-4 and DAdV-3 were 2.84 copies/µL and 2.85 copies/µL, respectively. A repeatability test demonstrated that the coefficient of variation was less than 2.5 % in both the intragroup and the intergroup analyses. Field sample distributions of FAdV-4 and DAdV-3 were investigated, and the percentages of DAdV-3-positive, FAdV-4-positive and coinfection-positive in Muscovy ducks were 27.78 %, 16.67 % and 11.11 %, respectively. Further studies are needed to provide more insight into the pathogenesis of FAdV-4 and DAdV-3 coinfection in ducks. In conclusion, the qPCR-HRM assay provides an accurate, sensitive, reliable and cost-effective alternative method for detecting and distinguishing FAdV-4 and DAdV-3.

Development and evaluation of a lateral flow immunochromatographic assay for the rapid detection of donkey meat in beef as a tool for meat adulteration identification

Development and evaluation of a lateral flow immunochromatographic assay for the rapid detection of donkey meat in beef as a tool for meat adulteration identification

Comparison of RT.PCR and ELISA Methods in the Diagnosis of Human Cytomegalovirus in Kidney Transplant Patients

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing age- related CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran.

Quantitative MODS-Wayne assay for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis from sputum samples.

PZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.

Abstract A011: Binding of the exon junction complex shapes the landscape of m6A in RNA - a possible source of m6A dysregulation in cancer

Abstract N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic mRNA. Increasing evidence shows that m6A is involved in the biological functions of cancer cells, including proliferation, invasion, metastasis, and drug resistance. Dysregulation of m6A deposition and its associated machinery, including writers, erasers and readers, is observed in multiple cancer types, and the dysregulation profiles have potential as diagnostic, prognostic and/or predictive biomarkers. In healthy cells, m6A modifications are installed co-transcriptionally by the writer enzyme METTL3, resulting in an enrichment of m6A in the 3’UTR of genes. For many years, the mechanistic origin for the m6A enrichment in the 3’UTR was unclear. Recently, the Gregory lab and others have developed a model where the exon junction complex (EJC) plays a key role in directing m6A deposition by hindering access of METTL3 to short exons and exon/intron junctions. In this study, we build on this model using a novel, proximity-barcoding based assay (EpiPlex™) for the simultaneous detection of m6A and inosine. Working with HEK293T cells, we modulated the activity of a central component of the EJC, the RNA helicase EIF4A3. Both genetic knockdown and chemical inhibition of EIF4A3 resulted in increased m6A levels and more diffuse localization at the 3’UTR. New m6A sites appear on short exons and near exon/intron junctions. These data corroborate the necessity of EIF4A3 for EJC assembly and confirm the model that the EJC prevents METTL3 from accessing and methylating exon junctions. Additionally, we find that blocking of the exon junction is reliant on EIF4A3’s helicase activity. Although the role of EJC in the cell cycle and cancer are still investigational, it is likely that m6A dysregulation is induced by aberrant EJC function, which presents a potential molecular mechanism for the dysregulation of multiple genetic pathways in cancer progression. Citation Format: Andrew Price, Erdem Sendinc, Byron Purse, Richard I Gregory, Gudrun Stengel. Binding of the exon junction complex shapes the landscape of m6A in RNA - a possible source of m6A dysregulation in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A011.

Abstract B013: An mDETECT assay for monitoring treatment response in metastatic breast cancer patients

Abstract Metastatic breast cancer treatment response is currently assessed every 3-6 months and this long delay results in many patients undergoing ineffective and toxic treatments for long periods of time. We have developed the methylation DETEction of Circulating Tumour DNA (mDETECT) assay, a targeted DNA methylation-based Next Generation Sequencing liquid biopsy that detects cancer-specific patterns in all subtypes of breast cancer. The assay targets 60 differentially hypermethylated regions in breast cancer across the genome and assesses over 400 individual CpGs with an average of 8 CpGs per target amplicon. The assay has been assessed on DNA from 73 breast cancer tumour samples which on average had 40 regions hypermethylated per patient with similar rates across all subtypes. We will present results from plasma samples from locally advanced and metastatic breast cancer patients along with plasma from age-matched, healthy female controls. A prospective, multi-centre observational cohort study to monitor metastatic breast cancer patients using the mDETECT liquid biopsy as they undergo treatment is active and recruiting patients. Blood is drawn with every standard-of-care blood draw for up to 3 years. Plasma has been assessed using the mDETECT assay to determine the methylation status of the DNA at each time point. 35 patients have been enrolled to date with ongoing recruitment up to 150 patients. Patients are being monitored with the mDETECT assay for earlier detection of disease progression on a treatment. They will also be monitored through treatment changes to assess a patient’s initial response to a given treatment. Changes in mDETECT levels will be correlated with clinical response to determine if the mDETECT breast cancer assay can detect a response to treatment at an earlier timepoint. This assay could allow for improved monitoring of treatment response allowing clinicians to make informed decisions, improving outcomes and prolonging patient’s lives. Citation Format: Keira Frosst, Brooke Wilson, Katrina Cristall, Catherine Crawford-Brown, Christopher R Mueller. An mDETECT assay for monitoring treatment response in metastatic breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B013.

Abstract A047: Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique

Abstract There is a continues need to develop technologies to detect and characterize circulating tumor cells that can become aggressive, metastatic, and hard to treat. Among these aggressive CTC are cells known as Hypoxic cell populations that learn to adapt and survive to low oxygen levels. Our project aims to test the applicability of a novel technique to detect Hypoxic Circulating Tumor Cells (H-CTC) that can move from tumors to circulating blood invading other tissues and becoming resistant to chemotherapy and radiation. The proposed assay incorporates a set of patented organic compounds as markers of hypoxic circulating tumor cells (H-CTC) with potential clinical applications. To date, our research has generated preliminary in-vitro studies with a novel NBQ-TOM demonstrating its capacity as markers of hypoxic human cancer cells in vitro. Comparison with the control Pimonidazole demonstrated the advantages of novel NBQ- TOM compounds as hypoxic marker. To determine the capacity of NBQ-TOM as hypoxic fluorescent marker, samples of 15 ml human whole blood were spiked with hypoxic or normoxic colon cancer cells (COLO 205). Spiked hypoxic or normoxic cells are then enriched, recovered, and treated with NBQ-TOM (25uM) for 24 hours. After treatment generated fluorescence is measured with an OPTIMA BMG Fluorimeter to demonstrate the significant formation of a fluorescent metabolite on hypoxic tumor cells in contrast to cells treated under normoxic environment. Also, fluorescence microscopy analysis confirmed the stronger fluorescence generation at hypoxic conditions. These preliminary results strongly support the applicability of these molecules for an in-vitro detection assay of hypoxic CTC in circulating blood that can become metastatic. Additional sensitivity and specificity analysis needs to be implemented. Citation Format: Beatriz Zayas, Karoline Rios, Osvaldo Cox. Detecting Hypoxic Circulating Tumor Cells (H-CTC) with a novel technique [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A047.

Abstract A065: Estimation of HPV16-induced cervical cancer cells in routine Thin-prep cytology samples of cancer patients

Abstract Persistent infection with human papillomavirus has been implicated in the etiology of cervical cancer and sustained expression of its E7 oncoprotein has been rendered suggestive of malignant transformation. However, there are limited E7 protein-based detection assays. This might be due to paucity of data on steady-state expression level of E7 protein and the number of HPV-induced cervical cancer cells in routine Thin-prep cytology samples. So, the objective of the present study was to identify and estimate HPV16-induced cervical cancer cells in cytology samples of cancer patients, with the goal to generate a quantitative immunological method in the cytological diagnosis of cervical lesions. We collected 25 Thin-prep cytology samples of cervical cancer patients (PAP test confirmed) from Obstetrics and Gynecology department, AIIMS, New Delhi. Samples were conditioned to enrich cellular components and were then quantified using hemocytometer for total cell count, PAP staining for cancerous cell count and immunofluorescence using HPV16 E7 antibody for HPV16-induced cancerous cell count. The method devised efficiently found an average number of 7 × 103 cells/ ml to be E7 positive, out of total 27 x 104 cells/ ml. Although PAP test detected 71 x 103 cells/ ml to be abnormal or cancerous, only 10% of them were found to be HPV16 transformed, suggesting HPV E7 as the primary marker for detecting transformed cells and thereby, identifying patients at-risk and prompting a better prognosis. This simple and efficient method deduced allows optimizing the number of HPV16-induced cervical cancer cells from cytology samples which directly correlates to the amount of HPV-16 major oncoprotein- E7 expression, and the disease severity. Citation Format: Srishty Raman, Pranay Tanwar, Neerja Bhatla, Subhash Chandra Yadav. Estimation of HPV16-induced cervical cancer cells in routine Thin-prep cytology samples of cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A065.

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12–0.21% and 0.62–1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

Laboratory validation of a clinical metagenomic next-generation sequencing assay for respiratory virus detection and discovery.

Tools for rapid identification of novel and/or emerging viruses are urgently needed for clinical diagnosis of unexplained infections and pandemic preparedness. Here we developed and clinically validated a largely automated metagenomic next-generation sequencing (mNGS) assay for agnostic detection of respiratory viral pathogens from upper respiratory swab and bronchoalveolar lavage samples in <24 h. The mNGS assay achieved mean limits of detection of 543 copies/mL, viral load quantification with 100% linearity, and 93.6% sensitivity, 93.8% specificity, and 93.7% accuracy compared to gold-standard clinical multiplex RT-PCR testing. Performance increased to 97.9% overall predictive agreement after discrepancy testing and clinical adjudication, which was superior to that of RT-PCR (95.0% agreement). To enable discovery of novel, sequence-divergent human viruses with pandemic potential, de novo assembly and translated nucleotide algorithms were incorporated into the automated SURPI+ computational pipeline used by the mNGS assay for pathogen detection. Using in silico analysis, we showed that after removal of all human viral sequences from the reference database, 70 (100%) of 70 representative human viral pathogens could still be identified based on homology to related animal or plant viruses. Our assay, which was granted breakthrough device designation from the US Food and Drug Administration (FDA) in August of 2023, demonstrates the feasibility of routine mNGS testing in clinical and public health laboratories, thus facilitating a robust and rapid response to the next viral pandemic.

Validation of the Level 2 Modification for the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method for Detection of the Monophasic Variant Salmonella enterica 1,4,[5],12:-:1,2.

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method, Performance Tested Method SM (PTM) 122302, is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately. The objective of the study was to validate the MDA2-SE/ST method for detection of an additional monophasic variant of ST, S. enterica 1,4,[5],12:-:1,2. A single strain of S. enterica 1,4,[5],12:-:1,2 was tested as part of a seven-strain inclusivity/exclusivity test panel. Strains were tested after growth in two enrichment media used in the method, buffered peptone water (BPW) and buffered peptone water-ISO formulation (BPW-ISO), and at two incubation temperatures, 35 ± 2 °C and 41.5 ± 1 °C. S. enterica 1,4,[5],12:-:1,2 produced positive results in the ST assay and negative results in the SE assay in both enrichment media and at both incubation temperatures. Results for the other six test strains were as expected under all conditions. The MDA2-SE/ST method can detect the monophasic variant S. enterica 1,4,[5],12:-:1,2 as well as S. enterica ser. Typhimurium and the monophasic variant S. enterica 1,4,[5],12:i:-. The MDA2-SE/ST method maintains inclusiveness for S. enterica ser. Typhimurium despite flagellar antigen variation. The data were reviewed by the AOAC PTM Program and approval was granted for modification of the MDA2-SE/ST method, PTM 122302.

Validation of the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method for Specific Detection of Salmonella enterica Ser. Enteritidis and Salmonella enterica Ser. Typhimurium in Chicken Carcass Rinse and Raw Ground Chicken AOAC Performance Tested MethodSM 122302.

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a rapid, nucleic acid amplification-based test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4 , [5] , 12: i:-, in select poultry samples. Results for SE and ST are generated separately. The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in chicken carcass rinse and raw ground chicken (325 g) for Performance Tested Methods SM (PTM) certification. The study consisted of inclusivity/exclusivity testing and independent laboratory testing of chicken carcass rinse and raw ground chicken using inoculated matrixes. Data were analyzed using a paired probability of detection model to determine if differences in the number of positive results obtained with the MDA2-SE/ST and reference methods were significant. In inclusivity testing, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1 Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, results for the MDA2-SE/ST and reference methods were in complete agreement for both matrixes. Results of the validation study showed that the MDA2-SE/ST method is an accurate, specific method for detection of SE and ST in select poultry matrixes. The data were reviewed by the AOAC PTM Program and approval was granted for certification of the Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium method as PTM 122302.

Level 2 Modification (Matrix Extension) Study of the Neogen® Molecular Detection Assay 2 - Salmonella Enteritidis/Salmonella Typhimurium Method: Detection of Salmonella enterica ser. Enteritidis and Salmonella enterica ser. Typhimurium in Cooked Breaded Chicken and Raw Ground Chicken (25 g).

The Neogen® Molecular Detection Assay 2-Salmonella Enteritidis/Salmonella Typhimurium (MDA2-SE/ST) method is a nucleic acid amplification test for specific detection of Salmonella enterica ser. Enteritidis (SE) and Salmonella enterica ser. Typhimurium (ST), including the ST monophasic variant Salmonella enterica 1,4,[5],12:i:-, in select poultry samples. Results for SE and ST are generated separately. The method was previously validated for testing of chicken carcass rinse and raw ground chicken (325 g). The objective of the study was to validate the MDA2-SE/ST method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g) as a Level 2 modification for Performance Tested Methods SM certification. Inclusivity/exclusivity testing and independent laboratory testing of cooked breaded chicken and raw ground chicken (25 g) were conducted. In inclusivity testing with BPW-ISO broth at 41.5 °C, all 50 SE strains produced positive results in the SE assay and negative results in the ST assay. All 53 ST strains (including the monophasic variant) produced positive results in the ST assay and negative results in the SE assay. All 35 exclusivity strains produced negative results in both assays. The exclusivity panel included multiple non-SE group D1 Salmonella serovars, multiple non-ST group B serovars, Salmonella spp. from other somatic groups, and other Enterobacteriaceae. In matrix testing, there were no significant differences in the number of positive results obtained with the MDA2-SE/ST and reference methods by probability of detection analysis at the 95% confidence level. Results of the matrix extension study showed that the MDA2-SE/ST method is an accurate method for detection of SE and ST in cooked breaded chicken and raw ground chicken (25 g). Validation of the MDA2-SE/ST method for two additional matrixes provides users with additional capability for specific detection of SE and ST in poultry products.

A comparison of two MALDI-TOF MS based assays for the detection of carbapenemases in Enterobacterales

Carbapenem resistant (CRE) and carbapenemase producing Enterobacterales (CPE) in particular, represent a major threat for healthcare systems worldwide. Rapid, reliable, and easy to perform assays are required to enable targeted and effective therapy. MALDI-TOF MS based carbapenemase diagnostics has potential advantages over molecular and phenotypic sensitivity tests, especially in terms of time to result. So far, only one mass spectrometry (MS)-based carbapenemase test system is commercially available for routine use. The aim of this study was to compare the performance of the established system to a novel MS-based test to identify CPE isolates. Forty consecutive CRE isolates (70% CPEs) were pre-screened for carbapenemase activity by routine laboratory methods. Isolates then were tested using the to date only IVD CE certified MALDI-TOF MS carbapenemase detection assay (MBT STAR-Carba IVD Kit; Bruker Daltonics) and a novel test designed for the recently launched EXS2600 MALDI-TOF MS system (Carbapenemase Activity Kit; Zybio). Valid results were obtained for 93% and 85% isolates by the Bruker and the Zybio assay, respectively. Sensitivities, specificities, positive and negative predictive values were 92%, 91%, 96%, and 83% for the Bruker assay and 96%, 64%, 85%, and 88% for the Zybio assay. There are notable differences concerning the handling of the assays. In summary, both systems featured high sensitivities for the detection of carbapenemases, but the Bruker assay yielded less false-positive results. There are advantages and disadvantages concerning the handling for each system, but both proved to be suitable for the use in a routine laboratory.

Establishment of a duplex TaqMan-based real time RT-PCR assay for simultaneous detection of BRSV and BVDV

Bovine respiratory disease complex (BRDC) represents a global acute respiratory condition that imposes substantial economic burdens on the cattle industry due to its high morbidity and mortality rates. Various factors contribute to the development of BRDC, including pathogen infections, environmental stresses, weaning of calves, and herd relocation. Viral pathogens, notably bovine respiratory syncytial virus (BRSV) and bovine viral diarrhea virus (BVDV), play a critical role in the etiology of BRDC, with single or combined viral infections being particularly clinically significant. In this study, we developed a duplex TaqMan-based real-time RT-PCR assay targeting the conserved regions of the F gene of BRSV and the 5′ UTR sequence of BVDV. The limits of detection for BRSV and BVDV were 6.83 copies/μL and 5.24 copies/μL, respectively. Our validation data suggest the assay has excellent sensitivity, specificity and reproducibility. Testing of clinical samples revealed prevalence of BRSV and BVDV in local farms in Jiangsu Province, China. This study provides an efficient diagnostic tool for the epidemiological investigation of BRDC.

Quantitative immunochromatographic assay for rapid detection of rosemary herb extract in ham sausage and dairy products

Quantitative immunochromatographic assay for rapid detection of rosemary herb extract in ham sausage and dairy products

Development of a LAMP assay for the rapid visual detection of the emerging tick-borne Songling virus

BackgroundSongling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established.MethodsIn this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay.ResultsA SGLV-specific LAMP assay was established with a detection limit of 1 × 10–2 copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples.ConclusionsWe successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV.Graphical

A novel lateral flow assay for lead ion detection based on G-quadruplex

A novel lateral flow assay for lead ion detection based on G-quadruplex

Validation of a molecular multiplex assay for the simultaneous detection and differentiation of bluetongue virus and epizootic haemorrhagic disease virus in biological samples.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Andalusia, Spain in 2022, and soon after in Portugal, and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR pan-BTV/pan-EHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity and specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation, and can be a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.

Identification of new antigenic epitopes of porcine reproductive and respiratory syndrome virus nsp12 protein using monoclonal antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arteritis virus family, significantly impacts the swine industry due to its high infectiousness. nsp12, the nonstructural protein encoded by PRRSV, is a membrane-associated protein, with limited knowledge about its antigenic properties and functions. In this study, we used the expressed and purified nsp12 protein as antigen to immunize mice, and successfully screened five positive hybridoma cell lines that stably secrete anti-nsp12 monoclonal antibodies using immunological assays such as indirect ELISA and IFA. The antigenic epitopes recognized by the five monoclonal antibodies were identified using the fusion expression of peptides derived from the overlapping truncators of the nsp12 gene. The results showed that monoclonal antibodies 3G11 and 9C2 recognized the antigenic epitope 93TWGFESDTAY102, 2E3 recognized 115DYNDAFRARQ124, and 10G6 and 2A4 recognized 142PGPVIEPTL150. Furthermore, the three newly identified antigenic epitopes were all immunodominant and located on the surface of nsp12 protein. Notably, the antigenic epitopes 93–102 aa are all highly conserved across PRRSV-2 strains, making them suitable targets for PRRSV-2 detection. In conclusion, our findings advance the understanding of the antigenic properties of the PRRSV nsp12 protein and facilitate the development of assays for PRRSV detection.