One-step Reverse Transcription-polymerase Chain Reaction Research Articles

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qampmini) with a preprogrammed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces high false positive results. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies) and fast results (less than 120 minutes), which would be suitable for field testing applications. Our high detection sensitivity platform provides fast and accurate results in 120 minutes without doing DNA/RNA extraction.

Coinfection With Severe Fever With Thrombocytopenia Syndrome and Scrub Typhus in Korea.

Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the 2 most common tick-borne infectious diseases in Korea. Every year, an increasing number of cases are reported, which is a public health concern. Therefore, we aimed to investigate the prevalence of SFTS-scrub typhus coinfection in patients with SFTS. Clinical samples were collected from 129 patients with SFTS. One-step reverse-transcription polymerase chain reaction (PCR) was performed to identify the SFTS virus (SFTSV), and real-time PCR followed by nested PCR was performed to detect the Orientia tsutsugamushi gene for scrub typhus. Phylogenetic analysis was conducted to confirm the evolutionary relationships among different species. Among 129 SFTS cases, 2 patients with SFTSV were positive for O. tsutsugamushi with a prevalence of coinfection of 1.6% (95% confidence interval, .001-.06). Phylogenetic analysis confirmed these as O. tsutsugamushi strain Boryong. Our study found that 1.6% of patients were coinfected with SFTS and scrub typhus infection. We believe that this information will add a new dimension to clinical diagnosis, which should be considered for better public health management. Further research is needed to better understand the ecological transmission dynamics and geographical distribution of SFTSV and O. tsutsugamushi in endemic countries.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Examination of the utility of the COVID-19 detection kit, TRC Ready® SARS-CoV-2 i for nasopharyngeal swabs.

The reverse transcription polymerase chain reaction (RT-PCR) offers high sensitivity, but has some drawbacks, such as the time required for the RNA extraction. Transcription reverse-transcription concerted reaction (TRC) Ready® SARS-CoV-2 i is easy to use and can be performed in about 40 minutes. TRC Ready® SARS-CoV-2 i and real-time one-step RT-PCR using the TaqMan probe tests of cryopreserved nasopharyngeal swab samples from patients diagnosed with COVID-19 were compared. The primary objective was to examine the positive and negative concordance rates. A total of 69 samples cryopreserved at -80° C were examined. Of the 37 frozen samples that were expected to be RT-PCR positive, 35 were positive by the RT-PCR method. TRC Ready® SARS-CoV-2 i detected 33 positive cases and 2 negative cases. One frozen sample that was expected to be RT-PCR positive was negative on both TRC Ready® SARS-CoV-2 i and RT-PCR. In addition, one frozen sample that was expected to be RT-PCR positive was positive by the RT-PCR method and negative by TRC Ready® SARS-CoV-2 i. Of the 32 frozen samples that were expected to be RT-PCR negative, both the RT-PCR method and TRC Ready® SARS-CoV-2 i yielded negative results for all 32 samples. Compared with RT-PCR, TRC Ready® SARS-CoV-2 i had a positive concordance rate of 94.3% and a negative concordance rate of 97.1%. TRC Ready® SARS-CoV-2 i can be utilized in a wide range of medical sites such as clinics and community hospitals due to its ease of operability, and is expected to be useful in infection control.

Survival outcomes and prognostic factors of lung cancer patients with the MET exon 14 skipping mutation: A single-center real-world study.

The MET exon 14 skipping (METex14) mutation is an important oncogenic driver in lung cancer. We performed a retrospective analysis of clinical data from lung cancer patients with the METex14 mutation to analyze their survival outcomes and associated prognostic factors. A one-step reverse transcription-polymerase chain reaction to examine the presence of the METex14 mutation was performed using RNA samples from 1374 lung cancer patients with no detected EGFR and ALK mutations. Pathological features and immunohistochemistry (IHC) results for c-MET were analyzed in patients with METex14-positive tumors. METex14 was identified in 69 patients with lung cancer, including 53 adenocarcinoma (ADC) and 16 non-ADC patients. In comparison with patients without the METex14 mutation, lung cancer patients harboring the METex14 mutation were generally elderly individuals, never-smokers, and had poor performance scores. A higher frequency of METex14 mutations was detected in pulmonary sarcomatoid carcinoma (PSC) patients (24.3%, n = 9/37). However, stage IV PSC patients with or without the METex14 mutations showed similarly poor overall survival (OS) (p = 0.429). For all 36 METex14-positive lung ADCs, multivariate analysis showed several poor prognostic factors, including strong c-MET IHC staining (p = 0.006), initial brain metastasis (p = 0.005), and administration of only supportive care (p < 0.001). After excluding seven patients who received only supportive care, we further analyzed 29 stage IV lung ADC patients with METex14 mutations who received anti-cancer treatment. Multivariate analysis showed that pemetrexed treatment (p = 0.003), lung radiotherapy (p = 0.020), initial brain metastasis (p = 0.005), and strong c-MET IHC staining (p = 0.012) were independent prognostic factors for OS in these patients. A higher frequency of METex14 mutations was detected in PSC patients. Stage IV PSC patients with or without the METex14 mutations had similarly poor overall survival. Pemetrexed-based chemotherapy, strong c-MET ICH staining, initial brain metastasis, and lung radiotherapy, may help predict survival outcomes in patients with advanced lung ADCs harboring the METex14 mutation.

Sensitivity and specificity of a rapid antigen assay for the detection of SARS-CoV-2 during the surge of B.1.1.529 variant

Background: As COVID-19 spread across the globe, the new variants continued to emerge, leading to a rapid surge in the cases and thereby overwhelming the testing laboratories. As Rapid antigen test kits became available, they complimented the reverse transcription polymerase chain reaction (RT-PCR) based testing and thereby moderately disburdening the testing laboratories. However, to increase the testing capacities, the regulatory authorities approved these rapid kits with little scrutiny. Therefore, validation of rapid antigen testing kits becomes essential before they are used as viable alternatives to RT-PCR based testing. Aims and Objectives: The aim of this study was to compare the sensitivity and specificity of OSKIT SARS-CoV-2 corona antigen test (Oscar Medicare Pvt. Ltd., India) to that of COVID-19 ONE-STEP RT-PCR KIT (Meril Diagnostics Pvt. Ltd., India). Materials and Methods: In this prospective study, spanning the peak of third wave in India (Between December 1 and January 31, 2022), 242 specimens were collected and analyzed by comparing their results both by OSKIT SARS-CoV-2 Corona Antigen Test and COVID-19 ONE-STEP RT-PCR KIT. The various clinico-epidemiological attributes of the patients were taken into consideration and used to analyze the results. Results: Rapid antigen tests (RAT) was positive in 44 individuals, giving an overall sensitivity of 67.31%. Sensitivity increased when Ct value of below 30 was taken a positive or when only symptomatic individuals were taken for analysis. Sensitivity was highest in case the duration of symptoms was &lt;5 days (92.86%). When comparing RAT results with Ct value of the screening gene (N gene), a higher proportion of positive cases was observed for lower Ct values. Further analysis revealed that majority of the RAT positive cases were from that subset of symptomatic patients who had a history of symptoms of &lt;5 days. Conclusion: The COVID-19 rapid antigen test evaluated in this study was able to detect SARS-CoV-2 infection with high viral loads in both asymptomatic and symptomatic individuals. Thus, this test can serve as a rapid tool for reducing community spread of the virus. The study concludes that the duration since symptom onset greatly affects sensitivity of antigen testing.

A chemiluminescence based approach to nucleic acid testing to detect HBV, HCV, and HIV-1 in blood screening

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) are life-threatening blood-borne infections that can be unintentionally transmitted in transfused blood products. Here, we optimized a method for detecting HBV, HCV, and HIV-1 in blood screening using magnetic nanoparticles (MNPs) and polymerase chain reaction (PCR)-chemiluminescence. Viral genomes were extracted from donated blood samples using MNPs. The isolated viral DNA and RNA were amplified in a one-step parallel reverse transcription-PCR (RT-PCR). HBV, HCV, and HIV-1 were detected using complementary nucleic acid probes and quantified using a chemiluminescent substrate. 10 422 donated blood samples were tested both with the above method and Roche Cobas TaqScreen MPX Test. The lengths of the amplified PCR products for HBV, HCV, and HIV-1 were 119 bp, 220 bp, and 174 bp, respectively, indicating that the extraction methods successfully separated high-quality viral genomes from the serum samples. The probes and reaction conditions used for the chemiluminescent detection of HBV, HCV, and HIV-1 genomes in unknown samples were empirically optimized. Of the 10 422 blood samples screened, 12 were HBV positive and 2 were HCV positive. These results were consistent with those of a parallel-controlled study using Roche Cobas TaqScreen MPX Test. The assay described here is fast, accurate, and sensitive for detecting HBV, HCV, and HIV, which runs in parallel and has broad implications for blood screening and epidemiological studies.

Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction.

The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

Screening of West Nile Virus, Herpesvirus, and Parvovirus in Rattus spp. in Klang Valley, Malaysia

Free-roaming and scavenging lifestyles of Rattus spp. in densely populated urban areas expose them to multiple pathogens that facilitate the transmission of infection to the human population more rapidly, raising public health concerns. There is limited information on the status of rat susceptibility to virus infection, particularly West Nile virus (WNV), herpesvirus, and parvovirus, to prepare for emerging zoonosis. A total of 23 (n = 23) blood samples collected from Rattus spp. in the wet market areas of Klang Valley, Malaysia, were subjected to molecular assay using a one-step reverse transcription-polymerase chain reaction (RT-PCR) to detect the highly conserved region of the WNV capsid and pre-membrane protein via nested polymerase chain reaction (PCR) assay targeting highly conserved amino acid motifs within the herpesviral DNA-directed DNA polymerase gene (DPOL) and polymerase chain reaction (PCR) assay targeting the parvovirus non-structural (NS) protein. As a result, 4 out of 23 (17.39%) rats were positive for herpesvirus DNA, but none were positive for WNV RNA and parvovirus DNA. The positive PCR amplicons of herpesvirus DNA were subjected to partial DNA sequencing analysis, 100% identical to Acomys herpesvirus SVMS 226,222 from Betaherpesvirinae, which is highly suggestive of rat cytomegalovirus (RCMV). This study has successfully demonstrated the presence of RCMV from Rattus spp. in the Klang Valley. The RCMV potentially crosses species barriers and establishes infection, raising public health concerns. The non-viraemic state of WNV or parvovirus infection, low sample size, and limited niche distribution emphasise the need for the expansion of this study in the future.

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

A survey of viruses and viroids in astringent persimmon (<i>Diospyros kaki</i>Thunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

Fabrication of an Oscillating Thermocycler to Analyze the Canine Distemper Virus by Utilizing Reverse Transcription Polymerase Chain Reaction.

The reverse transcription-polymerase chain reaction (RT-PCR) has been utilized as an effective tool to diagnose the infectious diseases of viruses. In the present work, the oscillating thermocycler is fabricated and performed to carry out the one-step RT-PCR process successfully. The ribonucleic acid (RNA) mixture is pipetted into the fixed sample volume inside an aluminum reaction block. The sample oscillates the pathway onto the linear motion control system and through the specific RT-PCR heating zones with individual homemade thermal control modules. The present oscillating thermocycler combines the merits of the chamber type and the CF type systems. Before PCR, the reaction chamber moves to the low-temperature zone to complete the RT stage and synthesize the complementary deoxyribonucleic acid (DNA). Next, the low-temperature zone is regulated to the annealing zone. Furthermore, the reactive sample is moved back and forth among three isothermal zones to complete PCR. No extra heating zone is required for the RT stage. The total length of the moving displacement of the chamber is within 100 mm. The miniaturization of the oscillating thermocycler can be expected. In our oscillatory device, the denaturation zone located between the annealing and extension zones is suggested as the appropriate arrangement of the heating blocks. Heat management without thermal cross-talk is easy. Finally, an improved oscillating device is demonstrated to execute the RT-PCR process directly, utilized to amplify the canine distemper virus templates successfully, which could be well applied to a low-cost DNA analysis system in the future.

Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse- transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV strains.

Development and evaluation of one-step real-time RT-PCR assay for improved detection of foot-and-mouth disease virus serotypes circulating in Egypt

Foot-and-mouth disease (FMD) is an extremely contagious and economically important viral disease affecting livestock. Rapid and precise diagnosis of FMD is of critical importance for efficient control and surveillance strategies of the disease. In this study, one-step real-time reverse transcription-polymerase chain reaction (RT-qPCR) assays were developed using newly designed primers/probe sets in the conserved regions within the VP1 coding sequence for specific detection of FMDV serotypes SAT 2 and O with their different lineage circulating in Egypt. The assays were validated for efficacy to detect different lineages of these endemic FMDV serotypes in Egypt; the detection limit was 10 genomic copies for serotype SAT 2 and one genomic copy for serotype O, with no cross-reactivity observed. These findings were confirmed by the specific and sensitive detection of FMDV in clinical samples obtained from different regions in Egypt and representing a range of subtypes within the SAT 2 and O serotypes. The results illustrated the potential of tailored RT-qPCR methods for the rapid detection and serotyping of FMDV belonging to different lineages of serotypes SAT 2 and O circulating in Egypt with high sensitivity and specificity. The developed assays could be easily deployed for routine surveillance and hence improving the disease control measures.

Quantification of hepatitis C virus in viremic blood donors in Iran: Need to reinforce post-donation follow up

IntroductionHepatitis C virus (HCV) infection is a public health problem and a major cause of chronic liver disease around the world. The main route of HCV transmission is contact with small quantities of infectious blood. Knowledge of the distribution of HCV viral load is essential to control HCV infection. This study aimed to investigate the HCV viral load distribution among Iranian blood donors. Materials and methodsThis cross-sectional study was conducted on 160 HCV confirmed blood donors with detectable HCV RNA who referred to blood transfusion centers for post-donation counseling all over the country. HCV RNA was quantified using an in-house one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) kit. Statistical analysis was performed in STATA version 13. ResultsThe mean age of the participants was 37.66 years. Out of 160 subjects, 156 (97.5 %) were male. The median viral load of the subjects was 7.7 × 104 (range: 2.28 × 10 3–3.42 × 107 IU/mL). Out of 160 blood donors, 70 (43.75 %, 95 % CI 0.36−0.51) had a viral load ≤5 × 104 IU/mL, and 90 (56.25 %, 95 % CI: 0.49−0.64) had a viral load >5 × 104 IU/mL. DiscussionThe distribution of HCV viral load among viremic blood donors emphasizes on the role of post-donation follow up in identification of blood donors potentially need for HCV anti-viral therapies.

RT-PCR test for specific indentification of influenzavirus (A/H5N1) in vaccine

RT-PCR (Reverse transcription - Polymerase Chain Reaction) is applied to determine the presence of influenza virus A/ H5N1 in vaccine, and to develop an identity process for specific virus strain A/H5N1 in influenza vaccine A/H5N1. Selected samples included: Ivacflu-A/H5N1 vaccine (Institute of Vaccines and Biologicals), Vaxigrip vaccine (Sanofi Pasteur), Influenza virus strain A/ Vietnam/1194/2004(A/H5N1) (NIBSC) was used as positive control; vaccine Varivax (MSD) and DNA/RNA free water was used as negative controls. The results showed that virus strain A/H5N1 was identified as production of RT-PCR that were positive with amplified primer pairs of 2 specific gene sequences of HA whose length 428 and 249 bp. Before starting RT-PCR, it was necessary to eliminate aluminum and the components of RT-PCR reaction included: 5X QIAGEN OneStep RT-PCR Buffer(5µl); dNTP (1µl); forward and reverse primers (1,5 µl); Enzyme (1 µl), H2 O (10 µl), ARN template (5 µl) and thermal cycle of RT- PCR reaction was: 50oC (30 minutes); 95OC (15 minutes); 94OC (30 seconds); 55OC (30 seconds); 72OC (1 minute); 72OC (10 minutes), 45 cycles.

Epidemiological characteristics and genetic diversity of norovirus infections among outpatient children with diarrhea under 5\xa0years of age in Beijing, China, 2011\u20132018

BackgroundHuman noroviruses are the leading cause of sporadic cases and outbreaks of viral acute gastroenteritis in all age groups worldwide.MethodsEpidemiological data and fecal specimens were collected between January 2011 and December 2018 from 4911 children < 5 years of age with diarrhea in three districts of Beijing. From 2011 to 2013, One-Step Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to detect noroviruses, and from January 2014 to December 2018, norovirus GI and GII were screened using duplex quantitative real-time RT-PCR (qRT-PCR). One-Step RT-PCR and RT-seminested PCR were performed to amplify the RNA-dependent polymerase and capsid genes of noroviruses in positive sample. Amplified products were sequenced directly; norovirus was typed using the online Norovirus Genotyping Tool v2.0 and phylogenetic analyses were conducted using MEGA-X.ResultsFrom 2011 to 2018, noroviruses were detected in 16.5% of specimens from children with diarrhea. The highest prevalence was observed in children aged 12 to 23 months (22.4%, 319/1421), followed by children aged 6 to 11 months (17.6%, 253/1441). The highest prevalence of norovirus infections occurred in autumn followed by winter, spring, and summer. From 2011 to 2018, the most prevalent dual types (genotype and polymerase type) were GII.4 Sydney[P31] (51.6%, 239/463), followed by GII.3[P12] (24.0%, 111/463), GII.4 2006b[P4 2006b] (7.3%, 34/463), GII.2[P16] (5.0%, 23/463), GII.17[P17] (2.6%, 12/463) and GII.6[P7] (2.6%, 12/463). GII.4 2006b[P4 2006b] predominated in 2011 and 2012. GII.4 Sydney[P31] predominated from 2013 to 2018. In total, 15 genotypes, 15 P-types and 19 dual types were detected in this study, reflecting the genetic diversity.ConclusionsThere were significant epidemiological characteristics and genetic diversity among outpatient children with norovirus infections < 5 years of age in Beijing from 2011 to 2018. These characteristics differ from those of norovirus outbreaks in Beijing. The complete genome sequences of each genotype are needed to better understand norovirus evolutionary mechanisms.

First report of Soybean dwarf virus infecting white clover (Trifolium repens) in Finland.

Soybean dwarf virus (SbDV, genus Luteovirus) is a single-stranded positive-sense RNA virus able to infect several legume species. SbDV was first reported in Japan where it was associated with significant yield losses in soybean (Tamada, 1969). Since then the virus has been detected worldwide. In Europe, the virus has only been reported from Germany (Abraham et al. 2007; Gaafar et al. 2020). In July 2018, several white clover plants (Trifolium repens L.) with leaf discoloration were observed in different locations in Oulu region in northern Finland. Individual plants were collected and analysed for the presence of viruses using small-RNA (sRNA) sequencing (Kreuze et. al. 2009) and reverse transcription-PCR (RT-PCR). Total RNA was extracted using EZNA micro RNA kit (Omega Bio-Tek, GA, USA). For sRNA analysis, sequencing libraries were constructed using the TruSeq small RNA library prep kit (Illumina, CA, USA) and sequenced on Illumina MiSeq platform. On average, 1.3 million single-end reads were obtained per sample, of which 27% were 18-25 nt long and used for the subsequent analysis. Contig assembly and virus identification with VirusDetect software (Zheng et al. 2017) detected SbDV in five out of six white clover samples analysed. Depending on the sample, 26-39 contigs (with lengths up to 301-469 nt) aligned to complete genome of a SbDV isolate previously described from white clover in USA (accession no. JN674402). The cumulative alignment coverage ranged from 35.5 % to 65.3 % with nucleotide identities between 94.4 % and 97.3 %. Additionally, two samples seemed to contain an unidentified closterovirus and one contained White clover cryptic virus 2. No additional viruses were detected from two of the samples.To confirm the presence of SbDV, the samples were tested by RT-PCR using primers MDF, MYF and MUR in multiplex (Schneider et al. 2011) together with SuperScript III One-Step RT-PCR System with the Platinum Taq DNA polymerase kit (Thermo Fisher Scientific, USA), essentially as instructed by the manufacturer. RT-PCR product of approximately 400 bp was produced from each of the five samples previously tested SbDV positive by sRNA analysis. No products were produced from the sample that was SbDV negative in sRNA analysis. Direct sequencing of two of the PCR products produced 347 and 361 bp sequences (GenBank: MZ355392 and MW929169) that were 95.7 % and 95.2 % identical, respectively, to a SbDV isolate (accession no. AB038148) that causes yellowing on soybean and is transmitted by Acyrthosiphon pisum (Terauchi et al. 2003). To our knowledge this is the first report of SbDV in Finland. SbDV is transmitted only by aphids (neither mechanical nor seed transmission occurs). In siRNA analysis all the isolates from Finland formed contigs that aligned almost perfectly (100 % coverage with ≥ 99 % nucleotide identity) to the coat protein (accession no. EF466131) of an SbDV isolate transmittable from white clover to faba bean by A. pisum (Abraham et al. 2007), an aphid common in Finland. Although significant yield losses by SbDV have only been reported on soybean (Tamada, 1969), the virus also causes symptoms in other legume crops, such as growth reduction on pea (Tian et al. 2017) and faba bean (Abraham et al. 2007), both of which are cultivated in Finland.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.