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Group A rotavirus (RVA) is the major foodborne pathogen that could be transferred via oysters. There are limitations of current methods for detecting rotavirus in oysters, such as the long duration of the traditional cell culture-...
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Group A rotavirus (RVA) is the major foodborne pathogen that could be transferred via oysters. There are limitations of current methods for detecting rotavirus in oysters, such as the long duration of the traditional cell culture-based plaque assay, the false positive due to the free RNA using RT-PCR-based molecular methods, and the complex process of RNA extraction from the food matrix. Therefore, we developed a PGM-mediated in situ capture reverse transcription quantitative polymerase chain reaction (ISC-RT-qPCR) assay to investigate the encapsulated RVA in oysters. In parallel comparing tests for the cell-cultured viral samples, the ISC-RT-qPCR exhibited 10-fold greater sensitivity and better accuracy of distinguishing the encapsulated RVA than RTqPCR. Furthermore, the ISC-RT-qPCR demonstrated a higher detection rate than RT-qPCR when applied to detect RVA in the artificially contaminated oyster. In practical application, the detection rates of RVA in oysters collected from four different farms were respectively higher by ISC-RT-qPCR (37.9%, 28.6%, 27.3%, and 0.0%) than by RT-qPCR (0.0%, 13.8%, 4.5%, and 0.0%), making the ISC-RT-qPCR a better assay for disease monitoring and control. Overall, all results indicate that ISC-RT-qPCR is a promising method for detecting encapsulated RVA in oysters and simplifies the steps of virus concentration, virus extraction with higher sensitivity and accuracy than the conventional RT-qPCR assay.
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Seit dem Jahr 2006 führt das Auftreten der Blauzungenkrankheit in Nordwest-und Zentraleuropa zu betrachtlichen wirtschaftlichen Schaden. Als first-line Diagnostik wird zunehmend die Reverse Transkription Real-time PCR (RT-qPCR) e...
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Seit dem Jahr 2006 führt das Auftreten der Blauzungenkrankheit in Nordwest-und Zentraleuropa zu betrachtlichen wirtschaftlichen Schaden. Als first-line Diagnostik wird zunehmend die Reverse Transkription Real-time PCR (RT-qPCR) eingesetzt, was eine sorgfaltige Evaluierung der verwendeten Methoden durch das durchführende Labor bedeutsam macht. Im Rahmen einer internen Validierung wurden vier kommerziell erhaltliche BTV RT-qPCR Screening kits und zwei publizierte Methoden verglichen. Es zeigten sichzum Teil deutliche Unterschiede in Reaktionskinetik, analytischer und diagnostischer Sensitivitat, Spezifitat, sowie in der Eignung derTestkits für die Anwendung bei ausgewahlten BTV-empfang-lichen Wirtsspezies. Nur zwei der kommerziellen Kits waren inallen untersuchten Parametern den technisch aufwendigeren Referenzmethoden ebenbürtig oder sogar überlegen und stellen daher eine Alternative zum BTV RT-qPCR Screening mit publizierten Methoden dar.
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Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern...
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Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern virus (APLPV), and the nepoviruses Tomato ringspot virus (ToRSV) and Cherry leafroll virus (CLRV). These viruses affect various stone fruits such as apricots, cherries, peaches, plums, and almonds. The goal of this work was to improve the RT-qPCR detection of PNRSV, PDV, and ApMV in addition to developing three new RT-qPCR assays for the detection of APLPV, ToRSV and CLRV. Primers for conventional RT-PCR as well as primers and probes for RT-qPCR assays were designed after aligning coat protein (CP) gene sequences of geographically diverse isolates with the corresponding CP gene sequences from the GenBank, targeting regions with 100% sequence identity. The efficiency of each RT-qPCR assay, as well as the intra-and inter-assay variability were determined. These conventional RT-PCR and RT-qPCR assays were validated using purified total RNAs from 221 trees from the USDA Clonal Germplasm Repository orchards. The data showed that more isolates were detected by RT-qPCR than by RT-PCR.
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A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce ne...
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A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce necrotic leaf curl virus (LNLCV) found in the Netherlands in 2011 and Motherwort yellow mottle virus (MYMoV) found in Korea in 2014. Primers for reverse transcriptase-PCR (RT-PCR) and RT-qPCR were designed with the aim of testing for the presence of virus in plant samples collected from the field. Both methods successfully amplified the target from infected samples but not from healthy control samples. The specificity of the CaTV assay was also checked against other known carrot viruses and no cross-reaction was seen. A comparative study between methods showed RT-qPCR was the most reliable method, giving positive results in samples where RT-PCR fails. Evaluation of the Ct values following RT-qPCR and a direct comparison demonstrated this was due to improved sensitivity. The previous published Torradovirus genus specific RT-PCR primers were tested and shown to detect CaTV. Also, virus transmission experiments carried out suggest that unlike other species of the same genus, Carrot torrado virus could be aphid-transmitted. (C) 2016 Elsevier B.V. All rights reserved.
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Sugarcane bagasse (SCB) pre-treated with laccase was used as substrate in two acidogenic phases (1 P and 2 P). In 1 P, the pretreatment conditions were statistically optimized (pH 4.0, 37 degrees C, 0.4 U of laccase/mL, 24.0 g SCB...
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Sugarcane bagasse (SCB) pre-treated with laccase was used as substrate in two acidogenic phases (1 P and 2 P). In 1 P, the pretreatment conditions were statistically optimized (pH 4.0, 37 degrees C, 0.4 U of laccase/mL, 24.0 g SCB/L and 120 min of reaction), which allowed obtaining 84% more H-2 (166.8 mL/L) than SCB in natura (90.4 mL/L). In 2 P, the liquid fraction of the 1 P reactors was diluted (90%) and more 108.5 ml H-2/L was obtained. Furthermore, the quantification of enzymes genes responsible for the hydrolysis (Cel) and H-2 production (Hyd) was carried out. An increase in the expression of both genes was observed from the phase of highest H-2 production rates in P1. In 2 P, the 1 P low genic expression was indicative of the autochthonous bacteria activity. The 2nd acidogenic phase strategy proved to be a promising novelty for the use of pre-hydrolyzed substrates with concomitant production of more value-added products. (c) 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
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The earliest possible detection of pathogens is of utmost importance in limiting the spread of contagious diseases, such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV-2). Alterna...
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The earliest possible detection of pathogens is of utmost importance in limiting the spread of contagious diseases, such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV-2). Alternative sample matrices must enable sensitive detection of these swine pathogens without the need for standard blood collection in pigs by venipuncture. Here, we investigate the potential use of GenoTube swabs for PCR detection of PRRSV and PCV-2 in individual blood and oral fluid swabs from pigs after experimental PRRSV infection. The results were compared with matched serum and pen-wise collected oral fluid. It was shown that PCV-2 detection in GenoTube blood swabs, oral fluid swabs and pen-wise oral fluid is at least as sensitive as in serum, while PRRSV detection is most sensitive in serum, followed by the blood swabs and oral fluids. A good correlation was observed between PCV-2 and PRRSV loads in serum and blood swabs, while there was little or no correlation between serum and oral fluids. A modified extraction protocol enabled further improvement of PRRSV RNA recovery from GenoTube swabs. Furthermore, we show that GenoTube swabs are suitable for long-term (up to 56 days) storage of specimens at room temperature without significant influence on PRRSV RNA stability. In conclusion, blood and oral fluid collected by GenoTube swabs are practical and reasonable sample matrices for PRRSV and PCV-2 detection by RTq-PCR and qPCR, respectively. However, it has to be kept in mind that viral loads in oral fluid do not accurately reflect those in serum.
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We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and witho...
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We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a downregulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression.We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our 'zero-step' RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.
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Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultra...
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Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C (q) shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.
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Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2'-hydroxyl-group of ribonucleotides (2'-O-methylation) has been fo...
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Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2'-hydroxyl-group of ribonucleotides (2'-O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3' terminal 2'-O-methylation, it is difficult to monitor the dynamic change of 3' terminal 2'-O-methylation during various biological processes. Capitalizing on the finding that 3' terminal RNA 2'-O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2'-O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2'-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2'-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1).
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Triticale, a cross between rye and wheat, is a crop important for animal feed and the production of biogas and ethanol. Soil-borne viruses found in wheat and rye, such as Furoviruses and Bymoviruses, also infect triticale. In orde...
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Triticale, a cross between rye and wheat, is a crop important for animal feed and the production of biogas and ethanol. Soil-borne viruses found in wheat and rye, such as Furoviruses and Bymoviruses, also infect triticale. In order to evaluate resistance/tolerance it is necessary to accuratelyquantify virus content in the plants. We have developedRT-qPCR assays for the quantitative detection of the Bymovirus Wheat spindle streak mosaic Virus (WSSMV) and the Furovirus Soil-borne cereal mosaic virus (SBCMV) in fieldsamples. Both a SYBR Green and a hydrolysis probe approachwere tested. The RT-qPCR approach allows the quantitativeevaluation and differentiation of triticale resistance toWSSMV and SBCMV. The reproducible large-scale analysis of field samples is feasible.
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