Real-time RT-PCR Assay Research Articles

In vitro antioxidant and wound healing activity of Sargassum polycystum hydroethanolic extract in fibroblasts and keratinocytes

Objective: To investigate the in vitro antioxidant and wound healing properties of the hydroethanolic extract of Sargassum polycystum, and elucidate the mechanism of its wound healing activity. Methods: Human dermal fibroblast and HaCaT cells were used to evaluate the proliferation by sulforhodamine B and dsDNA assay after treatment with Sargassum polycystum extracts. Scratch wound healing and phalloidin-rhodamine staining were employed to observe migratory activity and filopodia formation, respectively. Western blot and real-time RT-PCR assays were performed to determine the protein and gene expressions related to wound healing activities. Results: The phytochemical analysis found a higher level of flavonoid than phenolic compound in Sargassum polycystumextracts. In human dermal fibroblast cells, Sargassum polycystum extracts at 50 and 100 µg/mL significantly increased fibroblast proliferation and the gene expressions of hyaluronic acid synthase 1 (HAS1), HAS2, HAS3, collagen type 1 alpha 1 chain (COL1A1), collagen type 3 alpha 1 chain (COL3A1), and elastin. The phosphorylation of Akt, ERK1/2, and p38 MAPK was also significantly upregulated after treatment with Sargassum polycystumextracts. Additionally, 50 and 100 µg/mL of the extracts prominently enhanced the proliferation, migration, and filopodia formation of HaCaT cells, as well as the protein levels of pFAK/FAK, pSrc/Src, pAkt/Akt, pERK1/2/ERK1/2, Rac1 and Cdc42. Conclusions: Sargassum polycystum extracts show promising wound healing activities in human dermal fibroblasts and keratinocytes.

A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses

Avian influenza viruses (AIVs) have caused a large number of epidemics in domestic and wild birds, and even posed a health challenge to humans. Highly pathogenic AIVs have attracted the most public attention. However, low pathogenic AIVs, including H4, H6, and H10 subtype AIVs, have spread covertly in domestic poultry, without obvious clinical signs. The emergence of human infections with H6 and H10 AIVs and the evidence of seropositivity of H4 AIV in poultry-exposed individuals indicated that these AIVs sporadically infect humans and could cause a potential pandemic. Therefore, a rapid and sensitive diagnostic method to simultaneously detect Eurasian lineage H4, H6, and H10 subtype AIVs is urgently required. Four singleplex real-time RT-PCR (RRT-PCR) assays were established based on carefully designed primers and probes of the conserved regions of the matrix, H4, H6, and H10 genes and combined into a multiplex RRT-PCR method to simultaneously detect H4, H6, and H10 AIVs in one reaction. The detection limit of the multiplex RRT-PCR method was 1–10 copies per reaction when detecting standard plasmids, and showed no cross-reaction against other subtype AIVs and other common avian viruses. Additionally, this method was suitable to detect the AIVs in samples from different sources, the results of which showed high consistency with virus isolation and a commercial influenza detection kit. In summary, this rapid, convenient, and practical multiplex RRT-PCR method could be applied in laboratory testing and clinical screening to detect AIVs.

Amplification-free CRISPR-Cas13a assay for detection of Taura syndrome virus

Taura syndrome virus (TSV), which causes Taura syndrome, poses serious threats to the aquaculture industry. To date, early detection is still an important way to control its epidemic and spread. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) 13a (CRISPR-Cas13a) system, has been used to detect virus RNA. In this study, we developed an amplification-free CRISPR-Cas13a assay for directly detection of TSV from shrimp RNA. This assay could detect 107 copies/μL (16.6 pM) TSV in vitro transcribed (IVT) RNA in 5 min and 106 copies/μL (1.66 pM) within 15 min at 37 °C. It could distinguish TSV RNA specifically without any cross reaction with other shrimp pathogens. 56 Penaeus vannamei clinical samples were validated using this newly established assay, revealing 100 % consistency with the typical real-time RT-PCR (RT-qPCR) assay. These results demonstrate that this new assay for TSV is accurate, rapid and cost-effective, thus, it is promising for early diagnosis of RNA virus in aquaculture.

Comparison of Pan-Lyssavirus RT-PCRs and Development of an Improved Protocol for Surveillance of Non-RABV Lyssaviruses.

Rabies is a zoonotic and fatal encephalitis caused by members of the Lyssavirus genus. Among them, the most relevant species is Lyssavirus rabies, which is estimated to cause 60,000 human and most mammal rabies deaths annually worldwide. Nevertheless, all lyssaviruses can invariably cause rabies, and therefore their impact on animal and public health should not be neglected. For accurate and reliable surveillance, diagnosis should rely on broad-spectrum tests able to detect all known lyssaviruses, including the most divergent ones. In the present study, we evaluated four different pan-lyssavirus protocols widely used at an international level, including two real-time RT-PCR assays (namely LN34 and JW12/N165-146), a hemi-nested RT-PCR and a one-step RT-PCR. Additionally, an improved version of the LN34 assay ((n) LN34) was developed to increase primer-template complementarity with respect to all lyssavirus species. All protocols were evaluated in silico, and their performance was compared in vitro employing 18 lyssavirus RNAs (encompassing 15 species). The (n) LN34 assay showed enhanced sensitivity in detecting most lyssavirus species, with limits of detection ranging from 10 to 100 RNA copies/µL depending on the strain, while retaining high sensitivity against Lyssavirus rabies. The development of this protocol represents a step forward towards improved surveillance of the entire Lyssavirus genus.

Human rapid influenza diagnostic test: efficacy and significance for using in animal

Rapid influenza diagnostic tests (RIDTs) could considerably assist in clinical management resolving outbreaks of diseases in humans and animals. The RIDT for animals is rarely available and considerably expensive, especially in Thailand. In this study, therefore, the objective of this study was to determine the capacity of a commercial human RIDT “QuickNavi™Flu2”, an RIDT for influenza viruses A and B, to detect influenza viruses A antigen including 2 inactivated swine influenza viruses: H1N1 and H3N2, canine parainfluenza virus, and feline calicivirus. In addition, the efficacy of QuickNavi™Flu2 was compared with the real-time polymerase chain reaction assay (real-time rt-PCR). Both of SIV were used as stock solutions, and were serially diluted 6 concentrations before testing. The stock and diluted solutions of both SIV, specimens of canine parainfluenza virus, feline calicivirus and 96 unknown samples were tested by QuickNavi™Flu2 according to the guideline. Then all of samples were repeated testing with real-time rt-PCR assay. Results showed that Quick Navi-Flu could not give positive results for dilutions lower than 1:10 and 1:100, respectively. The sensitivity and specificity values of QuickNavi™Flu2 test in comparison to real-time rt-PCR assay for influenza A virus were 36.1% and 100%, respectively. In conclusion, a human RIDT can detect swine influenza H1N1 and H3N2 in high viral concentration, and appeared to be a useful tool for screening in animals when the viral load is sufficient.

Dual Effects of miR-181b-2-3p/SOX21 Interaction on Microglia and Neural Stem Cells after Gamma Irradiation.

Ionizing radiation induces brain inflammation and the impairment of neurogenesis by activating microglia and inducing apoptosis in neurogenic zones. However, the causal relationship between microglial activation and the impairment of neurogenesis as well as the relevant molecular mechanisms involved in microRNA (miR) remain unknown. In the present study, we employed immunohistochemistry and real-time RT-PCR to study the microglial activation and miRNA expression in mouse brains. Real-time RT-PCR, western blot, ELISA, cell proliferation and cytotoxicity assay were used in BV2 and mouse neural stem cells (NSCs). In the mouse model, we found the acute activation of microglia at 1 day and an increased number of microglial cells at 1, 7 and 120 days after irradiation at postnatal day 3 (P3), day 10 (P10) and day 21 (P21), respectively. In cell models, the activation of BV2, a type of microglial cell line, was observed after gamma irradiation. Real-time RT-PCR analysis revealed a deceased expression of miR-181b-2-3p and an increased expression of its target SRY-related high-mobility group box transcription factor 21 (SOX21) in a dose- and time-dependent fashion. The results of the luciferase reporter assay confirmed that SOX21 was the target of miR-181b-2-3p. Furthermore, SOX21 knockdown by siRNA inhibited the activation of microglia, thereby suggesting that the direct interaction of 181b-2-3p with SOX21 might be involved in radiation-induced microglial activation and proliferation. Interestingly, the gamma irradiation of NSCs increased miR-181b-2-3p expression but decreased SOX21 mRNA, which was the opposite of irradiation-induced expression in BV2 cells. As irradiation reduced the viability and proliferation of NSCs, whereas the overexpression of SOX21 restored the impaired cell viability and promoted the proliferation of NSCs, the findings suggest that the radiation-induced interaction of miR-181b-2-3p with SOX21 may play dual roles in microglia and NSCs, respectively, leading to the impairment of brain neurogenesis.

Diagnostic value of urine qRT-PCR for the diagnosis of West Nile virus neuroinvasive disease

Short and low-level viremia and virorachia, antibody cross-reactivity, IgM persistence, and inaccessibility of neutralization test, make laboratory diagnosis of West Nile virus (WNV) infection difficult. Recent investigations imply that WNV is excreted in urine longer and at higher concentrations compared to blood. The detection of WNV nucleic acid in cerebrospinal fluid (CSF), serum, and urine samples collected from 41 patients with suspected WNV neuroinvasive disease, was done by real-time RT-PCR assay. CSF and serum samples were also serologically tested using anti-WNV IgM/IgG ELISA kits. WNV infection was confirmed in 46.3% of patients by positive WNV RNA results in serum and/or CSF samples. The WNV RNA testing of urine allowed confirmation of 31.7% more cases. No association between WNV RNA urine positivity and age, gender, or the day of sample collection was found. The urine qRT-PCR can be a valuable diagnostic test for confirmation of probable cases of WNV neuroinvasive disease.

An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5.

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.

High-throughput sequencing application in the detection and discovery of viruses associated with the regulated citrus leprosis disease complex.

Citrus leprosis (CiL) is one of the destructive emerging viral diseases of citrus in the Americas. Leprosis syndrome is associated with two taxonomically distinct groups of Brevipalpus-transmitted viruses (BTVs), that consist of positive-sense Cilevirus, Higrevirus, and negative-sense Dichorhavirus. The localized CiL symptoms observed in multiple citrus species and other alternate hosts indicates that these viruses might have originated from the mites and eventually adopted citrus as a secondary host. Genetic diversity in the genomes of viruses associated with the CiL disease complex have complicated current detection and diagnostic measures that prompted the application of High-Throughput Sequencing (HTS) protocols for improved detection and diagnosis. Two cileviruses are known to infect citrus, and among them only citrus leprosis virus C2 (CiLV-C2) hibiscus strain (CiLV-C2H) has been reported in hibiscus and passion fruit in the US. Based on our current CiL disease complex hypothesis, there is a high probability that CiL disease is associated with more viruses/strains that have not yet been identified but exist in nature. To protect the citrus industry, a Ribo-Zero HTS protocol was utilized for detection of cileviruses infecting three different hosts: Citrus spp., Swinglea glutinosa, and Hibiscus rosa-sinensis. Real-time RT-PCR assays were used to identify plants infected with CiLV-C2 or CiLV-C2H or both in mixed infection in all the above-mentioned plant genera. These results were further confirmed by bioinformatic analysis using HTS generated data. In this study, we utilized HTS assay in confirmatory diagnostics to screen BTVs infecting Dieffenbachia sp. (family: Araceae), Passiflora edulis (Passifloraceae), and Smilax auriculata (Smilacaceae). Through the implementation of HTS and downstream data analysis, we detected not only the known cileviruses in the studied hosts but also discovered a new strain of CiLV-C2 in hibiscus from Colombia. Phylogenetically, the new hibiscus strain is more closely related to CiLV-C2 than the known hibiscus strain, CiLV-C2H. We propose this strain to be named as CiLV-C2 hibiscus strain 2 (CiLV-C2H2). The findings from the study are critical for citrus growers, industry, regulators, and researchers. The possible movement of CiLV-C2H2 from hibiscus to citrus by the Brevipalpus spp. warrants further investigation.

Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.

Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction

BackgroundPeste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV.ResultsTo achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV.ConclusionSince the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.

Detection and Genomic Characterization of Bovine Rhinitis Virus in China.

Bovine rhinitis virus (BRV) is an etiological agent of bovine respiratory disease complex (BRDC) and can be divided into two genotypes-bovine rhinitis A virus (BRAV) and bovine rhinitis B virus (BRBV). However, knowledge about the prevalence and molecular information of BRV in China is still limited. In this study, 163 deep nasal swabs collected from bovines with BRDC syndrome on 16 farms across nine provinces of China were tested for BRAV and BRBV by a duplex real-time RT-PCR assay. The results showed that 28.22% (46/163) of the samples were BRV-positive, and the positive rates were 22.09% (36/163) for BRAV and 9.2% (15/163) for BRBV. The co-circulation of both BRV genotypes was observed on two farms. Furthermore, five near-complete BRV genomes, including three BRAVs and two BRBVs, were obtained. The phylogenetic analysis showed that the three obtained BRAVs were phylogenetically independent, while the two BRBVs exhibited significant genetic heterogeneity. Recombination analysis revealed that three BRAVs and one BRBV strain obtained in this study were recombinants. The present study confirmed the presence and prevalence of BRAV in China, and it found that both types of BRV are circulating in beef cattle, which contributes to a better understanding of the prevalence and molecular characteristics of BRV.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Evaluation of two SARS-CoV-2 IgG/IgM Rapid Tests in Capillary Blood Samples

As COVID-19 has spread rapidly around the world, it has become essential to detect the virus quickly and accurately for disease prevention and control. Therefore, in response to the COVID-19 pandemic, the need for rapid serological point-of-care test has increased. Recently, many antibody tests have been developed to detect IgM and/or IgG to SARS-CoV-2 in human blood. The authors conducted a prospective study to evaluate the performance of a rapid chromatographic immunoassay and a fluorescent immunoassay for the qualitative detection of specific antibodies, IgM and IgG to SARS-CoV-2 in capillary blood samples, compared to the real-time RT-PCR. The subjects included 70 patients who were confirmed positive by real-time RT-PCR and 70 people who were negative. STANDARD Q COVID-19 IgM/IgG Plus Test (chromatographic immunoassay) and Fluorescent immunoassay for IgM and IgG to SARS-CoV-2 (fluorescent immunoassay) were performed using capillary blood samples. Based on the results of real-time RT-PCR assay, clinical sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of two rapid tests were investigated. And the agreement rate between two rapid tests was also presented. Sensitivity, specificity, PPV and NPV of the chromatographic immunoassay were 82.9%, 98.6%, 98.3%, and 85.2%, respectively. At more than 7 days after the onset of symptoms, sensitivity increased to 87.3%. Sensitivity, specificity, PPV, and NPV were 81.4%, 100.0%, 100.0%, and 84.3%, respectively, for the fluorescent immunoassay. At more than 7 days after the onset of symptoms, sensitivity increased to 85.7%. The agreement rate of the two tests was 97.1%. STANDARD Q COVID-19 IgM/IgG Plus Test and STANDARD F COVID-19 IgM/IgG Combo FIA turned out very specific and sensitive enough to detect individuals infected to SARS-CoV-2. Also, these tests were simple, fast, visually interpretable, and required a small amount of capillary whole blood.

Improvement of two Mn2+ coordination polymers on cognitive function of aged rats after anesthesia

ABSTRACT Two Mn2+ coordination polymers (CPs) with the scientific terms of {[Mn(TTPA)·(H2O)2]·H2O} n (1) and {[Mn(TTPA)·(H2TTPA)]·2DMSO} n (2) were favorably created on the basis of multidentate linking organic ligand 2,5-bis-(1,2,4-triazol-1-yl)-terephthalic acid (H2TTPA) in the conditions of solvent-presupposed thermal reaction. To measure the influence of two Mn2+ coordination polymers with novel structures, the ELISA assay and real-time RT-PCR assay were conducted in this present research. First of all, the ELISA assay was conducted to measure the content of inflammatory cytokines released into the hippocampal tissue. In addition to this, the relative expression of the TAU protein in the brain was further determined with real-time RT-PCR assay.

Therapeutic effect of two transition metal coordination polymers on ovarian cancer by regulating the expression of estrogen receptor

ABSTRACT In the present study, via using a ligand featuring oxalamide groups N,N′-bis(4-phthalic acid) (H4L), two new Cu(II) and Co(II)-containing coordination polymers with the chemical formulae of [Cu2L(H2O)4]n (1) and [Co(H2L)(H2O)2]n (2) have been successfully prepared via reaction of the corresponding metal salts with the H4L ligand. The as-prepared two coordination polymers have been studied via the single crystal X-ray diffraction, elemental analysis, powder X-ray diffraction and thermogravimetric analysis. Their therapeutic effect and mechanism for ovarian cancer was evaluated and explored. Firstly, the inhibitory activity of the new compounds on the proliferation of the ovarian cancer was measured with CCK-8 assay after compound treatment. Besides, the relative expression of the estrogen receptor on the ovarian cancer cells after compound treatment was also determined with real-time RT-PCR assay.

Presence of SARS-CoV-2 on surfaces and materials in supermarket social areas in Türkiye

The aim of this study was to monitor the presence of SARS-CoV-2, the virus that cause the coronavirus disease 2019 (COVID-19), particularly on certain foods and surfaces that come in contact with food in district supermarkets in Ankara, Türkiye, where the highest number of COVID-19 cases was reported based on data from the Ministry of Health. For this purpose, a total of 172 samples were taken from 5 supermarkets in 4 districts in Ankara. RNA was extracted from the samples and RdRp gene-targeting reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were used to determine the presence of SARS-CoV-2. The results showed that all the supermarket samples collected during the period when there was a high number of COVID-19 cases in the district did not have SARS-CoV-2 except for one sample that was taken from a supermarket where COVID-19 had been detected among the staff. In this supermarket, COVID-19 RNA was detected with a high number of copies of 5 000, using Real-Time RT-PCR assay in pooled swab samples taken from salt shakers, pepper shakers, red pepper shakers, and vinegar and oil bottles in the social area that the staff used for lunchbreaks and other breaks. This finding shows that it is of great importance for public health agencies to monitor COVID-19 cases in food businesses in regions with a high number of cases and to take samples from these businesses at certain intervals, as a form of “early warning system.”

Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation.

Emergence of novel human pathogens pose significant challenges to human health as highlighted by the SARS-CoV-2 pandemic. Wastewater based epidemiology (WBE) has previously been employed to identify viral pathogens and outbreaks by testing samples from regional wastewater treatment plants. Near source tracking (NST) allows for more targeted WBE by analysing samples from individual buildings such as schools or even individual floors such as in multi-floor office buildings. Despite the public health advantages of WBE, few strategies exist for optimising NST sampling methodologies. Therefore, we developed a protocol to evaluate virus detection in NST sampling using Pepper Mild Mottle Virus (PMMoV) as a proxy for RNA viruses. PMMoV is the most abundant enteric human associated RNA virus and is present in peppers/pepper-containing foods. Two bespoke TaqMan RT-PCR assays were developed to detect a PMMoV genomic 5' region and a capsid associated gene. To evaluate the protocol against field samples, pepper homogenates were flushed down an in-use toilet (Liverpool School of Tropical Medicine, UK) to spike wastewater with PMMoV on multiple days, and samples collected from two sewage access points to validate NST samplers. These wastewater samples were assessed for PMMoV based on Ct values and results compared to pepper and Tabasco derived PMMoV positive controls. Positive detection of PMMoV was comparable and consistent in ten independent samples across two NST samplers regardless of pepper homogenate spiking. We have developed two novel one step TaqMan assays that amplify both PMMoV targets in viral RNA extractions from peppers, Tabasco, and wastewater samples with cDNA synthesis through to RT-PCR results taking approximately 30 minutes. Pepper homogenate flushing was not required to detect PMMoV in our wastewater samples, however this strategy of flushing PMMoV containing materials outlined here could be valuable in assessing and validating NST in buildings with no previous or current sewage flow.

Comparative Analysis of Tomato Brown Rugose Fruit Virus Isolates Shows Limited Genetic Diversity.

Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-22 locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.

In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through the entire world and has become the worst pandemic from December 2019 until now. The establishment of positive standards for molecular diagnostic testing for SARS-CoV-2 plays a critical role in development and assessment of diagnostic assays associated with the shortage of positive specimens and viral culture fluids. This study aims to establish a novel assay for in-vitro RNA fragment synthesis based on amplicons of self-priming PCR targeting envelope protein gene and RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. The cDNA library of the targeted genes of SARS-CoV-2 was generated by using long-primers named sMn1 forward/reverse primer (E gene) and sMn2 forward/reverse primer (RdRp gene) for self-priming PCR assays. The synthesised amplicons that overlap the target sequence of the World Health Organization (WHO) assay were cloned into a pGEM-T easy vector, then transformed into E. coli competent cells by conventional methods. The recombinant plasmids were used as materials for in-vitro RNA transcription. Concentrations of the in-vitro transcribed RNA were 200-800 ng/μl with A260/A280 ratios of 2.0-2.2. Gel electrophoresis showed a single band of each RNA molecule with sizes of 216 and 214 bases for sMn1- E and sMn2- RdRp gene, respectively. Furthermore, we effectively evaluated the in-vitro transcribed RNA by a one-step, real-time RT-PCR assay according to the standard WHO protocol. The stability of in-vitro RNA over a 6-month storage period was then investigated. In conclusion, our assay for in-vitro synthesis of RNA fragments transcribed from self-priming amplicons were successfully established and thus these positive standards were useful for molecular diagnosing of SARS-CoV-2.