scholarly journals Monitoring of rotavirus in treated wastewater in Tehran with a monthly interval, in 2017–2018

2020 ◽  
Vol 18 (6) ◽  
pp. 1065-1072
Author(s):  
Shadi Tavakoli Nick ◽  
Seyed Reza Mohebbi ◽  
Seyed Masoud Hosseini ◽  
Hamed Mirjalali ◽  
Masoud Alebouyeh
Keyword(s):  
Rna Extraction ◽  
Conventional Polymerase Chain Reaction ◽  
Treated Wastewater ◽  
Harsh Environments ◽  
Monthly Interval ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Wastewater Treatment Process ◽  
Polymerase Chain ◽  
Elution Method

Abstract Rotaviruses are among the major causes of viral acute gastroenteritis in newborns and children younger than 5 years worldwide. The ability of rotaviruses to remain infectious in harsh environments as well as in the wastewater treatment process makes them one of the most prevalent enteric viruses. The current study aimed to determine the presence of rotavirus genomes and to analyze them phylogenetically in secondary treated wastewater (TW) samples. In total, 13 TW samples were collected from September 2017 to August 2018. Viral concentration was carried out using the absorption-elution method, and after RNA extraction and cDNA synthesis, real-time and conventional polymerase chain reaction (PCR) were performed. A phylogenetic tree was drawn using Maximum Likelihood and Tamura 3-parameter using MEGA v.6 software. Rotavirus genomes were detected in 7/13 (53.8%) and 3/13 (23.07%) samples using reverse transcription (RT)-PCR and conventional PCR, respectively. Accordingly, phylogenetic analysis revealed G4P[8], G9P[4], and G9P[8] genotypes among the samples. The presence of rotavirus in secondary TW samples discharged into surface water emphasizes the importance of monitoring and assessing viral contamination in the water sources used for agricultural and recreational purposes.

scholarly journals Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽  
1997 ◽  
Vol 81 (2) ◽  
pp. 222-226 ◽  
Author(s):  
Donald J. MacKenzie ◽  
Morven A. McLean ◽  
Srima Mukerji ◽  
Margaret Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Woody Plants ◽  
Rna Extraction ◽  
Viral Rna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Apple Stem ◽  
Spin Column ◽  
Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

scholarly journals Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

2021 ◽  
Vol 14 (1) ◽  
pp. 144-154
Author(s):  
Nour H. Abdel-Hamid ◽  
Eman I. M. Beleta ◽  
Mohamed A. Kelany ◽  
Rania I. Ismail ◽  
Nadia A. Shalaby ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Roc Curve ◽  
Diagnostic Performance ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Serum Samples ◽  
Polymerase Chain ◽  
Pcr Targeting ◽  
Pcr Techniques

Background and Aim: Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real- Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera. Materials and Methods: One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard. Results: TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%. Conclusion: A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

scholarly journals A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates

2019 ◽  
Vol 11 (3) ◽  
pp. 135-140
Author(s):  
Sepideh Khodaparast ◽  
Ashraf Mohabati Mobarez ◽  
Mehdi Saberifiroozi
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Glutamate Dehydrogenase ◽  
Conventional Polymerase Chain Reaction ◽  
Conventional Pcr ◽  
Accessory Gene ◽  
Chain Reaction ◽  
Fecal Samples ◽  
Genotype 2 ◽  
Polymerase Chain

BACKGROUND Clostridium difficile is the major causative agent of nosocomial antibiotic-associated colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enterocolitis. METHODS Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS 57 (9.8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6.4%) colonies had tcdA+/tcdB+ genotype, 2 (0.3%) tcdA+/tcdB-, 4 (0.7%) tcdA-/ tcdB+ and the remaining 14 (2.4%) colonies were tcdA and tcdB negative. CONCLUSION These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

Detection of small round structured viruses in clinical and environmental samples by polymerase chain reaction

1995 ◽  
Vol 31 (5-6) ◽  
pp. 375-382 ◽  
Author(s):  
M. Wolfaardt ◽  
C. L. Moe ◽  
W. O. K. Grabow
Keyword(s):  
Polymerase Chain Reaction ◽  
Tap Water ◽  
Practical Method ◽  
Glass Wool ◽  
Polluted Water ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Elution Method

Norwalk (NV) and other small round structured viruses (SRSVs) have been identified as common causes of gastroenteritis. Outbreaks of Norwalk gastroenteritis have been associated with contaminated drinking water and food such as oysters and salads. The cloning and sequencing of the NV genome has made it possible to detect NV and related viruses by the reverse transcription-polymerase chain reaction (RT-PCR). We applied RT-PCR to detect SRSVs in faecal specimens from two gastroenteritis outbreaks in South Africa, designated “Christmas” and “Grootbrak” and were able to detect SRSVs in all of the three specimens from the Christmas outbreak and in two of 16 specimens from the Grootbrak outbreak. The RT-PCR procedure used appeared to be more sensitive for the detection of SRSVs in patient stool specimens than immune electron microscopy and NV antigen detection by enzyme linked immunosorbent assay. The RT-PCR procedure proved suitable for the detection of SRSVs in seeded samples of sewage, sewage sludge, river water, and tap water. However, sensitivity was lower for seeded samples of sewage and sludge than for tap water, which indicates interference by high levels of organic matter. The RT-PCR procedure was also used to show that small numbers of SRSVs can successfully be recovered from large volumes of water by means of a glass wool adsorption-elution method. Since no practical method is available for quantitation of the small numbers of SRSVs concerned, it was not possible to evaluate the efficiency of recovery. Although no SRSVs have been detected by direct testing of sewage and sludge samples, the results obtained in this study show that RT-PCR detection of SRSVs in sewage and polluted water environments is feasible, and that small numbers of the viruses can, like many other enteric viruses, successfully be recovered by means of a glass wool adsorption-elution method.

scholarly journals QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES

2015 ◽  
Vol 57 (4) ◽  
pp. 299-303 ◽  
Author(s):  
Rodrigo STAGGEMEIER ◽  
Marina BORTOLUZZI ◽  
Tatiana Moraes da Silva HECK ◽  
Fernando Rosado SPILKI ◽  
Sabrina Esteves de Matos ALMEIDA
Keyword(s):  
Water Samples ◽  
Conventional Polymerase Chain Reaction ◽  
Quantitative Methodology ◽  
Human Beings ◽  
Environmental Matrices ◽  
Conventional Pcr ◽  
Human Adenoviruses ◽  
Causative Agents ◽  
Polymerase Chain ◽  
Artesian Wells

SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

scholarly journals SARS-CoV-2 RNA detection in nasal mid-turbinate swabs

2020 ◽  
Author(s):  
Laura Dioni ◽  
Benedetta Albetti ◽  
Federica Rota ◽  
Valentina Bollati
Keyword(s):  
Rna Extraction ◽  
Positive Control ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Detection ◽  
Nasal Swabs ◽  
Internal Positive Control ◽  
Polymerase Chain ◽  
Genomic Regions

Abstract In this protocol, we describe a method to investigate the presence of SARS-CoV-2 RNA in nasal swabs, using a commercially available high-throughput Real-Time Polymerase Chain Reaction (RT-PCR) assay (TaqPath™ Covid-19 kit, ThermoFisher Scientific) in a 384-Well Plate. After Viral RNA extraction with QIAamp Viral RNA Mini kit, reverse transcription and cDNA amplification, all samples are assessed for the presence of three specific SARS-CoV-2 viral genomic regions and an internal positive control (IPC), in one Multiplex RT-PCR reaction.

scholarly journals An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

2021 ◽  
Vol 44 (3) ◽  
Author(s):  
Anis Afifah ◽  
Prachumporn Nounurai ◽  
Rejeki Siti Ferniah ◽  
Hermin Pancasakti Kusumaningrum ◽  
Dyah Wulandari ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Extraction Yield ◽  
Economic Feasibility ◽  
Rt Pcr ◽  
Rna Integrity ◽  
Guanidinium Thiocyanate ◽  
Sample Amount ◽  
Polymerase Chain ◽  
Yield And Purity

One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications.

scholarly journals Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioanna Smyrlaki ◽  
Martin Ekman ◽  
Antonio Lentini ◽  
Nuno Rufino de Sousa ◽  
Natali Papanicolaou ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Plaque Assay ◽  
Diagnostic System ◽  
Heat Inactivation ◽  
Rt Pcr ◽  
Viable Option ◽  
Chain Reaction ◽  
Clinical Patient ◽  
Major Bottleneck ◽  
Polymerase Chain

Abstract Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

scholarly journals Lessons from applied large-scale pooling of 133,816 SARS-CoV-2 RT-PCR tests

2021 ◽  
Vol 13 (589) ◽  
pp. eabf2823 ◽  
Author(s):  
Netta Barak ◽  
Roni Ben-Ami ◽  
Tal Sido ◽  
Amir Perri ◽  
Aviad Shtoyer ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Group Testing ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Open Questions ◽  
Assay Performance ◽  
Theoretical Predictions ◽  
Polymerase Chain ◽  
The Impact

Pooling multiple swab samples before RNA extraction and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to reduce costs and increase throughput of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests. However, reports on practical large-scale group testing for SARS-CoV-2 have been scant. Key open questions concern reduced sensitivity due to sample dilution, the rate of false positives, the actual efficiency (number of tests saved by pooling), and the impact of infection rate in the population on assay performance. Here, we report an analysis of 133,816 samples collected between April and September 2020 and tested by Dorfman pooling for the presence of SARS-CoV-2. We spared 76% of RNA extraction and RT-PCR tests, despite the frequently changing prevalence (0.5 to 6%). We observed pooling efficiency and sensitivity that exceeded theoretical predictions, which resulted from the nonrandom distribution of positive samples in pools. Overall, our findings support the use of pooling for efficient large-scale SARS-CoV-2 testing.

scholarly journals Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control

2006 ◽  
Vol 96 (11) ◽  
pp. 1223-1229 ◽  
Author(s):  
Giorgio Gambino ◽  
Ivana Gribaudo
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Simultaneous Detection ◽  
Grapevine Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Arabis Mosaic Virus ◽  
Polymerase Chain

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10-3 or 10-4 extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

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