Multiplex Reverse Transcription Research Articles

Development and Application of a Multiplex Reverse Transcription–Droplet Digital PCR Assay for Simultaneous Detection of Hepatitis A Virus and Hepatitis E Virus in Bivalve Shellfish

Foodborne viruses are significant contributors to global food safety incidents, posing a serious burden on human health and food safety. In this study, a multiplex reverse transcription–droplet digital PCR (RT-ddPCR) assay based on the MS2 phage as a process control virus (PCV) was developed to achieve the simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in bivalve shellfish. By optimizing the reaction system and procedures, the best reaction conditions were selected, and the specificity, sensitivity, and reproducibility of the method were assessed. Additionally, the MS2 phage’s recovery rate was utilized as an indicator to evaluate the optimal sample nucleic acid enrichment method. The results indicated that the RT-ddPCR assay exhibited optimal amplification efficiency with primer concentrations of 900 nmol/L, probe concentrations of 350 nmol/L for HAV and HEV, and 500 nmol/L for MS2, an annealing temperature of 53.1 °C, an extension time of 90 s, and 45 cycles. Additionally, the developed multiplex RT-ddPCR assay demonstrated high specificity, with quantitation limits of 12.6, 8.9, and 7.8 copies/reaction being observed for HAV, HEV, and the MS2 phage, respectively. A total of 240 bivalve samples were analyzed, of which 4 were positive for HAV and 12 for HEV. The viral loads for HAV ranged from 3048 to 6528 copies/2 g, while those for HEV ranged from 3312 to 20,350 copies/2 g. This assay provides a vital tool for enhancing food safety monitoring.

Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry.

Strawberry viruses are significant pathogenic agents in strawberry. The development and application of efficient virus detection technology can effectively reduce the economic losses incurred by virus diseases for strawberry cultivators. In order to rapidly identify strawberry virus species and prevent the spread of virus disease, a multiplex reverse transcription polymerase chain reaction system was established for the simultaneous detection and identification of strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry mottle virus (SMoV), strawberry polerovirus 1 (SPV-1), strawberry pallidosis-associated virus (SPaV), and strawberry crinivirus 4 (SCrV-4). In this study, six pairs of specific primers were designed on the conserved genomic regions of these viruses. The primer concentration, annealing temperature, and amplification cycle number of the reaction system were optimized. Subsequent sensitivity testing and application of the optimized detection system were carried out. The results indicate the establishment of an efficient detection system for strawberry viruses. The optimal reaction can detect the six viruses at the same time, which provides technical support for the early prevention and treatment of strawberry virus diseases.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses.

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

One-Step Multiplex RT-PCR Method for Detection of Melon Viruses.

This study presents a one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) method for the simultaneous detection of multiple viruses affecting melon crops. Viruses such as Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Tobacco mosaic virus (TMV), Papaya ring spot virus (PRSV), and Melon yellow spot virus (MYSV) pose a great threat to melons. The mixed infection of these viruses is the most common observation in the melon-growing fields. In this study, we surveyed northern Xingjiang (Altay, Changji, Wujiaqu, Urumqi, Turpan, and Hami) and southern Xingjiang (Aksu, Bayingolin, Kashgar, and Hotan) locations in Xinjiang province and developed a one-step multiplex RT-PCR to detect these melon viruses. The detection limits of this multiplex PCR were 103 copies/μL for ZYMV and MYSV and 102 copies/μL for WMV, SqMV, PRSV, CMV, and TMV. The detection results in the field showed 242 samples were infected by one or more viruses. The multiplex RT-PCR protocol demonstrated rapid, simultaneous, and relatively effective detection of viruses such as WMV, CMV, ZYMV, SqMV, TMV, PRSV, and MYSV. The technique is designed to identify these melon viruses in a single reaction, enhancing diagnostic efficiency and reducing costs, thus serving as a reference for muskmelon anti-virus breeding in Xinjiang.

Characterization of virus species associated with sweet potato virus disease in Costa Rica and riboprobe development for its rapid detection

AbstractSweet potato (Ipomoea batatas) is a crucial crop for food security and economic stability in many regions, including Costa Rica. This study aimed to assess the prevalence and genetic diversity of two major viruses associated with sweet potato virus disease (SPVD) in Costa Rica: sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). A total of 50 leaf samples displaying typical viral disease symptoms were collected from major sweet potato‐growing regions. Using multiplex reverse transcription (RT)‐PCR, SPFMV was detected in 82% of the samples and SPCSV in 52%, with a coinfection rate of 48%. Genetic analysis revealed that SPFMV isolates belonged predominantly to phylogenetic group B, while SPCSV isolates clustered with the West African (WA) strain. The low genetic diversity within these viral populations suggests a limited number of initial infection events followed by local spread. Riboprobe technology was evaluated as an alternative to RT‐PCR for virus detection. While the riboprobes for SPFMV demonstrated sensitivity comparable to RT‐PCR, the SPCSV riboprobes showed slightly lower sensitivity, particularly when using crude leaf extracts. Despite this, riboprobes offer significant advantages in terms of cost and ease of use for large‐scale screening. Our findings underscore the need for robust diagnostic and management strategies to control SPVD and highlight the importance of continuous monitoring and research to mitigate the impact of viral diseases on sweet potato production in Costa Rica. These insights contribute to the global understanding of SPVD and support collaborative efforts in plant pathology research.

Key performance evaluation of commercialized multiplex rRT-PCR kits for respiratory viruses: implications for application and optimization.

The complexity and severity of viral respiratory tract infections (RTIs) emphasize the pivotal role of precise diagnosis for viral RTIs in guiding effective public health responses and ensuring appropriate medical interventions, given the substantial population at risk. This study highlights the necessity and importance of evaluating the analytical validity of multiplex real-time reverse transcription polymerase chain reaction assays, offering valuable insights into their optimization and application.

Outbreak of severe acute respiratory infections caused by recombinant human adenovirus type B 7/3 in hospitalized infants from a nursery in Dakar, April 2024

ObjectivesAcute respiratory infections are among the leading cause of mortality in children under 5 years of age worldwide, with most of these deaths due to bronchiolitis and pneumonia. We investigated and analyzed a pediatric outbreak of acute respiratory infections that resulted in the hospitalization of four infants in a nursery in Dakar in late April 2024. MethodsNasopharyngeal specimens were collected from infants and tested for a panel of respiratory pathogens by multiplex real-time reverse transcription–polymerase chain reaction. Subsequently, the positive samples underwent next-generation sequencing for molecular analysis. ResultsHuman adenovirus (HAdV) was the principal etiologic agent detected in hospitalized infants with pneumonia and nearly half (46.7%; seven of 15) of the suspected cases identified during the investigation at the nursery. All the HAdV isolates were classified as being of subgroup B1. Molecular characterization revealed that infants attending the nursery were infected with a recombinant HAdV strain containing an adenovirus serotype 7 penton and serotype 3 hexon and fiber proteins. ConclusionsOur findings reinforce previous evidence that recombination leads to the emergence of new adenovirus strains with epidemic and lethal potential. These results emphasize the need to strengthen surveillance in inpatient settings across the country.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

Pestivirus bovis is implicated in repeated abortions and subfertility of vaccinated cattle in Egypt

The study of Pestivirus bovis, formerly known as Bovine Viral Diarrhea Virus (BVDV), is of significant importance due to the economic challenges it poses in cattle herds in several countries. This virus leads to decreased productivity, reproductive failures, and increased susceptibility to secondary infections. The current study aimed to investigate the role of BVDV in abortion and infertility among cattle in Egypt using advanced diagnostic techniques. We employed a one-step multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) to detect BVDV, and the results showed an overall prevalence of 11.2% among 178 tested samples. Notably, 11.2% tested positive for BVDV-1, with higher detection rates in adult cows (12.1%) compared to calves (9.85%). Importantly, 13.8% of samples from dams with subfertility and repeated abortions were positive for BVDV-1. The results of rRT-PCR guided virus isolation using Madin–Darby bovine kidney (MDBK) cells. Identifying cytopathic effects (CPEs) in 15% of the samples, consistent with cytopathic-BVDV (CP-BVDV). An indirect fluorescent antibody assay (IFA) confirmed the presence of CP-BVDV in these samples. For non-cytopathic (NCP)-strains, the immunoperoxidase (IP) test using bovine turbinate (BT) cells was more effective, detecting NCP-BVDV in samples without CPE in MDBK cells. The presence of mixed infection was indicated by an isolate from a diarrheic calf, showing positive results for both CP-BVDV and NCP-BVDV. Plaque assays further confirmed CP-BVDV and NCP-BVDV isolation from mixed infections, highlighting the selective amplification of both biotypes. These findings underscore the significant role of BVDV-1 in reproductive disorders in cattle and the importance of employing comprehensive diagnostic methods for effective control and management strategies. Our study provides valuable insights that can guide future studies and improve herd health and productivity.

The Distribution of Respiratory Viral Pathogens Among the Symptomatic Respiratory Tract Infection Patients From Dhaka City in the Pre-COVID-19 Pandemic Era.

Rapid and accurate diagnosis is crucial for determining the etiology and, perhaps, effectively treating and preventing viral respiratory infections. A multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay⁠⁠⁠⁠⁠⁠⁠ was utilized to determine the prevalence of viral etiology in cases of acute respiratory tract infections (ARTIs). Outpatient department (OPD) and intensive care unit (ICU) patients with fever and respiratory symptoms were enrolled from December 2018 to April 2020. Nucleic acids were extracted from the respiratory tract samples using the SV Total RNA Isolation System (Promega Corporation, Madison, WI), and virus identification was performed using qRT-PCR assay (Fast Track Diagnostics {FTD} Respiratory Pathogens, Esch-sur-Alzette, Luxembourg). A total of 152 samples were collected from OPD and ICU. Among them, 32.23% (n = 49) of the patients were positive for at least one respiratory virus. From 49 infected cases, 42 had only a single viral pathogen, whereas seven had co-infections. Of the patients, 32.25% (30) in the OPD and 32.20% (19) in the ICU tested positive for the respiratory viral pathogen. Among the OPD patients, human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1were detected as predominant viruses (10.75%), followed by influenza virus (IFV) (8.6%), human rhinoviruses (HRVs) (6.45%), human parainfluenza viruses (HPIVs) (6.45%), respiratory syncytial virus (RSV) (3.22%), and adenovirus (2.15%). In ICU cases, HPIV and HRV were detected as predominant viruses (8.47% each), followed by HCoV (5.08%), human metapneumovirus (HMPV) (5.08%), influenza A virus (IAV) (3.38%), adenovirus (3.38%), and RSV (1.69%). This study highlighted the prevalence of respiratory viruses in both the community and hospital settings during pre-COVID-19, indicating a significant presence among patients in these environments.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam

ObjectiveThis study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation.ResultsThe rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Multiplex reverse transcription recombinase polymerase amplification combined with lateral flow biosensor for simultaneous detection of three viral pathogens in cattle

Bovine viral diarrhea virus (BVDV), bovine epidemic fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) cause respiratory symptoms in cattle. The absence of rapid, precise, and easily accessible diagnostic methods poses difficulties for herders and veterinary epidemiologists during outbreaks of major infectious animal diseases. Considering the mixed infection of viruses, a multiple-detection method, reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), was established to simultaneously detect the three pathogens. This technique is based on the specific binding of three differently labeled RT-RPA products (DNA sequences) to antibodies on the three test lines of the LFB, achieving multiplex detection through the presence or absence of coloration on the LFB test lines. The fluorescence values of the LFB test lines are recorded by a test strip reader. The mRT-RPA-LFB assay completes detection at a constant temperature of 41 °C within 33 min. The limits of detection (LODs) for BVDV, BEFV and BRSV were 2.62 × 101, 2.42 × 101 and 2.56 × 101 copies/μL, respectively. No cross-reactivity was observed with the other six bovine viruses. The developed method showed satisfactory intra- and inter-assay precision, and the average coefficients of variation were ranged from 2.92 % to 3.99 %. The diagnostic sensitivity and specificity were 98.11 % and 100 %, respectively, which were highly consistent with the RT-qPCR assay, and the kappa value was 0.988 (95 % confidence interval, CI). In general, the mRT-RPA-LFB assay has the potential to become a powerful tool for rapid screening of cattle diseases because of its advantages such as fast detection speed, convenient operation, strong specificity, and high sensitivity.

Clinical performance of the ImmuneMed Dengue NS1 Ag Rapid I test for the diagnosis of dengue fever: a diagnostic accuracy study

Background: Dengue virus (DENV) is transmitted by mosquitoes and is becoming a global threat owing to an increase in the number of cases and mortality, especially in low- and middle-income tropical countries. Rapid and easy-to-use diagnostic tests are required to differentiate dengue fever from other febrile illnesses. Methods: We evaluated the clinical performance of the ImmuneMed Dengue NS1 Ag Rapid I test (ImmuneMed, Inc.) using positive and negative sera collected from patients with confirmed DENV infection and healthy individuals, respectively. The AccuPower® ZIKV (DENV, CHIKV) multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assay (Bioneer) was used as the reference standard to confirm DENV infection. Results: One hundred DENV-positive and 161 DENV-negative samples were evaluated. Overall, the sensitivity and specificity were 100% (95% confidence interval [CI], 96%–100%) and 100% (95% CI, 98%–100%), respectively. The sensitivity and specificity were 100% for both DENV-1 and DENV- 2. The sensitivity was the same (100%) for sera collected < 3 days and ≥ 3 days from symptom onset. The performance of the ImmuneMed Dengue NS1 Ag Rapid I and realtime RT-PCR tests showed strong overall agreement. Conclusion: The ImmuneMed Dengue NS1 Ag Rapid I test was highly specific for DENV and as sensitive as RT-PCR. These findings suggest that the ImmuneMed Dengue NS1 Ag Rapid I test may be a useful point-of-care test for dengue fever.

Dengue Hemorrhagic Fever in Quang Nam Province (Vietnam) from 2020 to 2022-A Study on Serotypes Distribution and Immunology Factors.

Background: Dengue hemorrhagic fever (DHF) is the most prevalent and fastest-growing vector-borne disease globally, with symptoms ranging from mild to severe and, in some cases, fatal. Quang Nam province in Vietnam can serve as a model for dengue epidemiological study, as it is an endemic region for DHF with a tropical climate, which significantly constrains the health system. However, there are very few epidemiological and microbiological reports on Dengue virus (DENV) serotypes in this region due to the limited availability of advanced surveillance infrastructure. Aims of the study: This study aims to (1) assess the PCR positivity rates among hospitalized patients with clinical Dengue presentation; (2) identify the circulating DENV serotypes; and (3) assess the impact of secondary DENV infections on outbreak severity by detecting the presence of DENV-specific IgG antibodies in the plasma of DENV-infected patients. Materials and methods: Blood samples from patients clinically diagnosed with DHF and admitted to Quang Nam General Hospital (2020-2022) were analyzed. RNA extraction was performed using the NKDNA/RNAprep MAGBEAD kit, followed by Multiplex Reverse Transcription real-time Polymerase Chain Reaction (MLP RT-rPCR) for DENV detection and serotype identification. Positive samples were further tested for DENV-specific IgG antibodies using an enzyme-linked immunosorbent assay (ELISA). Results: The PCR positivity rate among hospitalized patients was approximately 68% throughout the study period. A significant shift in DENV serotypes was observed, with DENV-2 initially dominant and later giving way to DENV-1. IgG was detected in nearly half of the MPL RT-rPCR-positive samples, indicating secondary DENV infections. Conclusions: Our study highlights persistent dengue prevalence and dynamic shifts in DENV serotypes in Quang Nam province, emphasizing the need for improved diagnostic strategies and timely sample collection. The significant serotype shifts and the presence of IgG in hospitalized patients suggest potential severe outcomes from recurrent DENV infections, possibly linked to antibody-dependent enhancement (ADE) effect, underscoring the importance of advanced surveillance, vector control, vaccination campaigns, and public education to predict and prevent future DHF epidemics.

Epidemiological changes in respiratory pathogen transmission among children with acute respiratory infections during the COVID-19 pandemic in Kunming, China

BackgroundAcute respiratory infections are a leading cause of morbidity and mortality in children. However, studies on the prevalence of respiratory viruses among children with acute respiratory infections in Kunming, China, are lacking. Therefore, we aimed to investigate the epidemiological characteristics of respiratory pathogens among children with acute respiratory infections in Kunming during the coronavirus disease 2019 pandemic.MethodsNasopharyngeal swab samples were collected from 4956 children with acute respiratory infections at Yunnan Provincial First People’s Hospital between January 2020 and December 2022, patients with COVID-19 were excluded from the study. Multiplex reverse transcription polymerase chain reaction was used to detect respiratory pathogens.ResultsThe frequency of respiratory pathogens among children was significantly lower in 2020 than in 2021 and 2022. The following pathogens had the highest prevalence rates (in descending order) from 2020 to 2022: HRV > RSV > PIV > ADV > MP; HRV > RSV > HADV > PIV > MP and HRV > Mp > HADV > H3N2 > HMPV. The overall frequency of respiratory pathogens exhibited an inverted U-shape with increasing age among the children. Human bocavirus, human parainfluenza virus, and human respiratory syncytial virus were the dominant respiratory viruses in children aged ≤ 3 years, whereas Mycoplasma pneumoniae was the dominant respiratory pathogen in children aged > 3 years. HRV has the highest prevalence and is the main pathogen of mixed infection. The prevalence of the influenza A virus has decreased significantly, whereas HRSV and Mp are found to be seasonal.ConclusionsOur findings offer an objective evaluation of transmission dynamics and epidemiological shifts in respiratory pathogens during the coronavirus disease 2019 pandemic in Kunming, serving as a basis for informed decision-making, prevention, and treatment strategies.

First report of Triticum mosaic virus infecting oat crop in the United States.

Triticum mosaic virus (TriMV, genus Poacevirus, family Potyviridae) was first reported in 2006 (Seifers et al. 2008) to infect wheat, and since then, it has been established as a constraint for US wheat production (Byamukama et al. 2013). In the field, TriMV often exists as a coinfection with wheat streak mosaic virus (WSMV), and these two viruses interact synergistically to produce severe symptoms and greater yield loss (Byamukama et al. 2012; Tatineni et al. 2022). Both TriMV and WSMV are transmitted by wheat curl mites (Aceria tosichella Keifer) (McMechan et al. 2014). Wheat is the primary host reported for TriMV in the field, but Seifers et al. (2010) established oat, rye, barley, and several other cereals and grasses as hosts under controlled conditions. However, there are no documented cases of TriMV infecting oats in the field. Between 10-25 June, 2023, a total of 273 field oat plants showing foliar yellowing, yellow flecking, and streaking symptoms were collected from four different fields in Nebraska (Big Springs: 41.1029° N, 102.1451° W; Mead: 41.2292° N, 96.4938° W; Odell: 40.0459° N, 96.7984° W; Stumf: 40.5048° N, 101.4223° W). Total RNA was extracted using the MagMax Plant RNA Isolation kit (Thermo Fisher Scientific) and the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) (Mondal et al. 2023). Sample RNA was assayed with a single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) to determine presence of WSMV and TriMV. Out of 273 symptomatic oat plants, 254 (93.04%) tested positive for at least one virus. Out of total positive samples, 238 were positive for WSMV (93.70 %), 12 plants tested positive for both TriMV and WSMV (4.70%), and 4 plants were infected with TriMV alone (1.60%). As a secondary confirmation, amplified fragments from the TriMV single infection were gel purified using a gel extraction kit (QIAquick) and sequenced (Eurofins Genomics). The nucleotide sequences were analysed using the BlastN program, compiled, and edited in the BioEdit software (Hall 1999). Sequences were deposited in the NCBI GenBank database (accession number PP475806). Nucleotide BLAST searches of the target coat protein (CP) gene showed > 98% identity to the corresponding sequences in TriMV accession MK318274. For further validation, virus inoculum was prepared by grinding field-collected plant material from plants with only TriMV present in 20 mM sodium phosphate buffer, pH 7.0, and then mechanically inoculating two-week-old oats (cv. Shaw n=8) and wheat (cv. Sattler, n=8) plants. Three weeks post-inoculation, all the eight wheat plants exhibited mild yellowing and streaking symptoms, while oat plants did not show obvious foliar symptoms. All wheat and oat plants were further tested positive with DAC-ELISA (antibodies produced against TriMV CP at the USDA-ARS facility in Lincoln, NE) and with RT-PCR. The specific attribution of these symptoms to TriMV in oats is not possible as none produced prominent symptoms. Asymptomatic oat infection from symptomatic field-collected oat samples could be due to oat cultivar differences. Although the prevalence of TriMV in wheat has been established across the Great Plains of the United States, to our knowledge, this is the first report of TriMV infection in US oat fields. Our finding warrant further investigation into the incidence and impact of the virus in oat crop and its potential for serving as a asymptomatic virus reservoir.

Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.

Plasmodium vivax is the main causative agent of malaria in Panama. However, the prevalence of asymptomatic infections in the different endemic regions remains unknown. Understanding the epidemiological behavior of asymptomatic infections is essential for the elimination of malaria. This study aimed to determine the prevalence of asymptomatic malarial infections in one of the main endemic regions of Panama using multiplex real-time reverse transcription RT-MqPCR. A cross-sectional study was conducted in three communities in the Guna Yala Comarca. A total of 551 thick blood smears and their respective samples on filter paper were collected from volunteers of different ages and sexes from June 20 to 25, 2016. Infections by the Plasmodium spp. were diagnosed using microscopy and RT-MqPCR. All statistical analyses were performed using the R software. The average prevalence of asymptomatic infections by P. vivax in the three communities detected by RT-MqPCR was 9.3%, with Ukupa having the highest prevalence (13.4%), followed by Aidirgandi (11.1%) and Irgandi (3.3%). A total of 74 samples were diagnosed as asymptomatic infections using RT-MqPCR. Light microscopy (LM) detected that 17.6% (13/74) of the asymptomatic samples and 82.4% (61/74) were diagnosed as false negatives. A 100% correlation was observed between samples diagnosed using LM and RT-MqPCR. A total of 52.7% (39/74) of the asymptomatic patients were female and 85.1% (63/74) were registered between the ages of 1 and 21 years. Factors associated with asymptomatic infection were community (aOR = 0.38 (95% CI 0.17-0.83), p < 0.001) and age aOR = 0.98 (95% CI 0.97-1.00), p < 0.05); F = 5.38; p < 0.05). This study provides novel evidence of the considerable prevalence of asymptomatic P. vivax infections in the endemic region of Kuna Yala, representing a new challenge that requires immediate attention from the National Malaria Program. The results of this study provide essential information for the health authorities responsible for developing new policies. Furthermore, it will allow program administrators to reorient and design effective malaria control strategies that consider asymptomatic infections as a fundamental part of malaria control and move towards fulfilling their commitment to eliminate it.

Comparative Analysis of Viral Load and Cytokines during SARS-CoV-2 Infection between Pregnant and Non-Pregnant Women.

To better understand the vulnerabilities of pregnant women during the COVID-19 pandemic, we conducted a comprehensive, retrospective cohort study to assess differences in immune responses to SARS-CoV-2 infection between pregnant and non-pregnant women. Nasopharyngeal swabs and serum specimens from 90 pregnant and 278 age-matched non-pregnant women were collected from 15 March 2020 to 23 July 2021 at NewYork-Presbyterian Queens Hospital in New York City. Multiplex reverse transcription polymerase chain reaction, neutralizing antibody, and cytokine array assays were used to assess the incidence, viral load, antibody titers and profiles, and examine cytokine expression patterns. Our results show a lower incidence of SARS-CoV-2 infection in pregnant women compared with non-pregnant women. Pregnant women infected with SARS-CoV-2 exhibited a substantially lower viral load. In addition, the levels of both anti-spike protein receptor-binding domain IgG neutralizing antibodies and anti-N Protein IgG were elevated in pregnant women. Finally, cytokine profiling revealed differential expression of leptin across cohorts. These findings suggest that pregnancy is associated with distinct immune and virological responses to SARS-CoV-2 infection, characterized by lower infection rates, substantially lower viral loads, and enhanced antibody production. Differential cytokine expression indicates unique immune modulation in pregnant women.

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5’UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5′ UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

A Multiplex RT-PCR for the Detection of Three Viruses and One Viroid Infecting Hemp

Hemp ( Cannabis sativa L.) is an emerging industrial crop in the United States. In recent surveys of hemp growers, disease identification and management were determined to be significant priority areas for future research. From previous studies on disease identification in Western U.S. hemp production systems, hop latent viroid (HLVd), beet curly top virus (BCTV), Cannabis sativa mitovirus 1 (CasaMV1), and citrus yellow vein associated virus (CYVaV) were detected at high incidences. In the present study, a multiplex reverse transcription PCR protocol was developed to detect this viroid and viruses from hemp leaf samples simultaneously. Using the developed protocol, the multiplex reverse transcription PCR could detect each virus and viroid specifically in 10−1-diluted cDNA. The protocol was validated on 6 hemp metavirome leaf samples from field-grown hemp grown in 2019 and 23 uncharacterized leaf samples in 2021 from Colorado. Of the samples tested, 95% tested positive for CasaMV1 in 2021. This was greater than infections of BCTV (4%), HLVd (0%), and CYVaV (0%). Coinfections accounted for 4% of samples in 2021. This method offers sensitive, specific, and fast simultaneous detection of three viruses and one viroid from hemp, contributing to large-scale pathogen-free hemp certification schemes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .