Assays For Detection Of Virus Research Articles

A multiplex RT-qPCR assay for simultaneous detection of cucurbit viruses from individual whitefly and plant samples.

Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vectors many viruses leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection of viruses in the whiteflies before crop production. One such tool is the multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets in a single reaction and simultaneously quantify the levels of each target, with a detection limit of 100 copies per target. In this study, a multiplex RT-qPCR-based detection system capable of identifying one DNA virus and three RNA viruses in whiteflies: cucurbit leaf crumple virus (CuLCrV), cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV) was developed. To ensure the reliability of the assay, an internal gene control as the fifth target to monitor false-negative results was incorporated. This newly developed molecular diagnostic tool possesses several advantages. It can detect up to five desired targets from a single whitefly RNA sample, even at concentrations as low as 1 ng/µl. To evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids and in vitro transcribed RNA transcripts of the target viruses. We also assessed the specificity of the assay by including aphid-transmitted viruses and other viruses known to infect cucurbits. The diagnostic method successfully detected all five targets simultaneously and allowed for the quantification of up to 100 copies using a mixture of healthy? RNA and in vitro transcribed RNA. Our aim with this study was to develop a highly specific and sensitive one-step multiplex RT-qPCR system for the simultaneous detection of viruses transmitted by whiteflies in cucurbits. This system offers significant advantages for early detection, enabling prompt control measures to mitigate the further spread of viral infections and reduce yield losses. Additionally, we demonstrated the ability to simultaneously detect mixed viruses (CCYV, CYSDV, CuLCrV, and SqVYV) in individual whiteflies and quantify the number of viral copies carried by each whitefly. The multiplex RT-qPCR assay outperforms currently available techniques for detecting many samples at a given time and can be effectively utilized for early monitoring of plant viruses in individual whiteflies and symptomless plants.

A multiplex RT-qPCR assay for simultaneous detection of cucurbit viruses from individual whitefly and plant samples.

Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vectors many viruses leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection of viruses in the whiteflies before crop production. One such tool is the multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets in a single reaction and simultaneously quantify the levels of each target, with a detection limit of 100 copies per target. In this study, a multiplex RT-qPCR-based detection system capable of identifying one DNA virus and three RNA viruses in whiteflies: cucurbit leaf crumple virus (CuLCrV), cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV) was developed. To ensure the reliability of the assay, an internal gene control as the fifth target to monitor false-negative results was incorporated. This newly developed molecular diagnostic tool possesses several advantages. It can detect up to five desired targets from a single whitefly RNA sample, even at concentrations as low as 1 ng/µl. To evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids and in vitro transcribed RNA transcripts of the target viruses. We also assessed the specificity of the assay by including aphid-transmitted viruses and other viruses known to infect cucurbits. The diagnostic method successfully detected all five targets simultaneously and allowed for the quantification of up to 100 copies using a mixture of healthy? RNA and in vitro transcribed RNA. Our aim with this study was to develop a highly specific and sensitive one-step multiplex RT-qPCR system for the simultaneous detection of viruses transmitted by whiteflies in cucurbits. This system offers significant advantages for early detection, enabling prompt control measures to mitigate the further spread of viral infections and reduce yield losses. Additionally, we demonstrated the ability to simultaneously detect mixed viruses (CCYV, CYSDV, CuLCrV, and SqVYV) in individual whiteflies and quantify the number of viral copies carried by each whitefly. The multiplex RT-qPCR assay outperforms currently available techniques for detecting many samples at a given time and can be effectively utilized for early monitoring of plant viruses in individual whiteflies and symptomless plants.

Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D'Aguilar Viruses.

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D'Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968-1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Development of recombinase amplification assays for the rapid detection of infectious myonecrosis virus

Infectious myonecrosis virus (IMNV) has affected shrimp farming in many countries, such as northeastern Brazil and southeast Asia, and poses a serious threat to the global shrimp industry. Reverse transcription enzymatic recombinant amplification technology (RT-ERA) is a rapid DNA amplification assay with high specificity in isothermal conditions and has been widely applied to the pathogen’s detection. In this study, two novel ERA assays of IMNV, real-time RT-ERA and an RT-ERA combined with lateral flow dipsticks assay (RT-ERA-LFD), were developed and evaluated. The real-time RT-ERA assay could be carried out at 38–42 °C and had the highest end-point fluorescence value and the smallest Ct value at 41 °C. The brightness and width of the detection line were at a maximum at 39 °C and 30 min, and these conditions were selected in RT-ERA-LFD. Both real-time RT-ERA and RT-ERA-LFD produced positive results with IMNV standard plasmids only and showed no cross-reaction with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis disease (VpAHPND); white spot syndrome virus (WSSV); infectious hypodermal and hematopoietic necrosis virus (IHHNV); or Ecytonucleospora hepatopenaei (EHP). Meanwhile, we compared the sensitivities of nested RT-PCR, real-time RT-PCR, real-time RT-ERA, and RT-ERA-LFD. The sensitivities of real-time RT-ERA and RT-ERA-LFD were both 101 copies/μL. The detection sensitivities of nested RT-PCR and real-time RT-PCR were 100 and 102 copies/μL, respectively. As a result, two ERA assays were determined to be specific, sensitive, and economical methods for the on-site diagnosis of IMNV infection, showing great potential for the control of IMNV infections.

Evaluation of biofire filmarray meningitis/encephalitis assay for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens in the setting of a tertiary care hospital

Evaluation of biofire filmarray meningitis/encephalitis assay for detection of bacteria, viruses, and yeast in cerebrospinal fluid specimens in the setting of a tertiary care hospital

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Loop‐mediated isothermal amplification assay: A novel disease diagnostics tool in sweetpotato seed quality assurance

AbstractSweetpotato is an important food crop in global production. However, sweetoptato viruses pose a threat to sustainable agriculture and cause significant economic loss. More than 30 viruses have been reported to date, with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV) occurring frequently and in combination, causing sweetpotato virus disease. The detection of viruses directly from sweetpotato is still a challenge, and efforts are being made toward this front. Therefore, rapid detection of viruses is critical for effective control. Current diagnostic tests are not sufficiently sensitive to reliably detect viruses directly from sweetpotato and require expensive laboratory equipment and a high level of skilled experience. Loop‐mediated isothermal amplification (LAMP) is sensitive and specific for both DNA/RNA amplification; it is affordable and its characteristics make it potentially suitable for on‐site testing. LAMP assays have been developed for several sweetpotato viruses: SPCSV, SPFMV, and sweet potato leaf curl virus (and related sweepoviruses). Laboratory validation showed a 100% diagnostic sensitivity for all three viruses. The LAMP on‐site testing results were comparable to those of polymerase chain reaction and reverse transcriptase polymerase chain reaction confirmatory laboratory tests. Interlab validation and packaging into affordable kits will ensure high adoption in sub‐Saharan Africa. The LAMP assay can be used for field surveys and monitoring the phytosanitary status of pre‐basic seed production in quarantine or certification program. This guarantees pathogen‐free planting material enters the seed system. We discuss opportunities for LAMP as a diagnostic assay for the rapid detection of sweetpotato viruses and its adoption as a quality control system for planting materials.

Development of a quantitative NS1 antigen enzyme-linked immunosorbent assay (ELISA) for Zika virus detection using a novel virus-specific mAb

Viruses from the Flaviviridae family, such as Dengue virus (DENV), Yellow fever virus (YFV), and Zika virus (ZIKV) are notorious global public health problems. ZIKV emergence in Polynesia and the Americas from 2013 to 2016 raised concerns as new distinguishing features set it apart from previous outbreaks, including its association with neurological complications and heightened disease severity. Virus detection is impaired as cross-reactivity to other closely related orthoflaviviruses is common among commercially available diagnostic kits. While non-structural protein 1 (NS1) has been used as an early marker of DENV and West Nile virus (WNV) infection, little is known about NS1 expression during ZIKV infection. In the present work, we developed a NS1 capture ELISA using a novel ZIKV-specific monoclonal antibody to study NS1 expression dynamics in vitro in mosquito and human cell lines. While detectable in culture supernatants, higher concentrations of NS1 were predominantly cell-associated. To our knowledge, this is the first report of NS1 detection in human cells despite viral clearance over time. Tests with human samples need to be conducted to validate the applicability of NS1 detection for diagnosis, but overall, the tools developed in this work are promising for specific detection of acute ZIKV infection.

Repeat testing of mpox specimens with late CTs improves detection of potential false positive cases.

The mpox pandemic necessitated the rapid development of clinical assays for monkeypox virus detection. While the majority of mpox specimens have high viral loads with corresponding early cycle threshold (CT) values, reports have indicated some specimens with late CT values can represent false positive results. To mitigate this risk, the Centers for Disease Control and Prevention (CDC) published an advisory recommending repeat testing of all specimens with CT values ≥34. However, limited experimental data were available to support this specific cutoff. In this study, we examine whether a more conservative approach in which all specimens with CT values ≥29 are repeated would improve the detection of potential false positive results. Compared to the CDC algorithm, our approach identified an additional 20% (5/25) of potential false positive results. To assess the impact of this cutoff on laboratory workload, we modeled the expected increase in test volume and turnaround time (TAT) relative to the CDC method. Using a lower repeat threshold, test volume increased by 0.7% while mean TAT increased by less than 15 minutes. Overall, a lower threshold than recommended by the CDC for repeating late CT mpox specimens may reduce the number of false positives reported while minimally impacting testing volume and TAT.

Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome

The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.

Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/μL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.

Sequence optimized diagnostic assay for Ebola virus detection

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013–2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Development of Polyclonal Antibodies and a Serological-Based Reverse-Transcription Loop-Mediated Isothermal Amplification (S-RT-LAMP) Assay for Rice Black-Streaked Dwarf Virus Detection in Both Rice and Small Brown Planthopper.

Rice black-streaked dwarf virus (RBSDV) infects rice and maize, and seriously affects rice yields in main rice-producing areas. It can be transmitted via small brown planthopper (SBPH: Laodelphax striatellus Fallén). To more rapidly, sensitively, and highly throughput diagnose RBSDV in the wild condition, we first purified the recombinant His-CPRBSDV protein, and prepared the polyclonal antibodies against the His-CPRBSDV protein (PAb-CPRBSDV). Based on the PAb-CPRBSDV, we developed a series of serological detections, such as Western blot, an enzyme-linked immunosorbent assay (ELISA), and a dot immunoblotting assay (DIBA). Furthermore, we developed a serological-based reverse-transcription loop-mediated isothermal amplification assay (S-RT-LAMP) that could accurately detect RBSDV in the wild. Briefly, the viral genomic dsRNA together with viral CP were precipitated by co-immunoprecipitation using the PAb-CPRBSDV, then the binding RNAs were crudely isolated and used for RT-LAMP diagnosis. Using the prepared PAb-CPRBSDV, four serology-based detection methods were established to specifically detect RBSDV-infected rice plants or SBPHs in the wild. The method of S-RT-LAMP has also been developed to specifically, high-throughput, and likely detect RBSDV in rice seedlings and SBPHs simultaneously. The antiserum prepared here laid the foundation for the rapid and efficient detection of RBSDV-infected field samples, which will benefit for determination of the virulence rate of the transmission vector SBPH and outbreak and epidemic prediction of RBSDV in a rice production area.

A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Establishment of a p30-based lateral flow assay for African swine fever virus detection

African swine fever virus (ASFV) has continuously devastated the global pig industry. Viral persistence causes problems in large pig farms and kills small farms. Timely diagnostic tools play an important role in controlling outbreaks and minimizing losses. In this study, we developed a lateral flow assay to detect ASFV on-site. The VDRG® ASFV Ag Rapid Kit was established using two monoclonal antibodies (mAbs) against the p30 protein. The conjunction pad of the kit was coated with a mixture of the mAb and colloidal gold. This rapid kit was capable of detecting 11.5 ng of antigen and 0.16 HAD50 of virus from samples, in 20 min for the entire procedure. It passed cross-specific tests using common viruses that cause infectious diseases in pigs. ASFV was detected after 4 days in experimental infection in pigs by the kit. The specificity and sensitivity of the kit for clinical samples were 99.88% and 84.52% (93.8% for samples with a Ct value below 30), respectively. Finally, the kit can detect 100% positive herd outbreaks. The VDRG® ASFV Ag Rapid Kit presents a useful point-of-care tool for ASFV detection.

Down-Classification of Hepatitis C Virus Diagnostics: Implications for Screening and Diagnosis.

In November 2021, the United States Food and Drug Administration reclassified 2 types of hepatitis C virus (HCV) diagnostic tests (HCV antibody and HCV nucleic acid) from class III to class II, providing a less burdensome pathway to market for diagnostic companies. This down-classification is anticipated to facilitate innovation in HCV diagnostics, particularly for new point-of-care viral detection assays, and ultimately support HCV elimination efforts by increasing the ease of screening as well as test-and-treat models of HCV care.

Laboratory assessment of a multi-target assay for the rapid detection of viruses causing vesicular diseases.

The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses.

Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).