scholarly journals Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

2005 ◽  
Vol 71 (7) ◽  
pp. 3734-3740 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Ronald Italiaander ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Large Volume ◽  
Water Samples ◽  
Extraction Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Virus Rna ◽  
Nasba Assay

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

scholarly journals Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

2006 ◽  
Vol 72 (8) ◽  
pp. 5349-5358 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Water Samples ◽  
Rna Extraction ◽  
Sewage Treatment ◽  
Rdrp Gene ◽  
Nucleic Acid Sequence ◽  
Viral Gastroenteritis ◽  
Rt Pcr ◽  
Inhibitory Factors

ABSTRACT Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

scholarly journals Detection of Schmallenberg Virus RNA in Bull Semen in Poland

2016 ◽  
Vol 19 (3) ◽  
pp. 655-657 ◽  
Author(s):  
J. Kęsik-Maliszewska ◽  
M. Larska
Keyword(s):  
Extraction Method ◽  
Rna Extraction ◽  
Viral Rna ◽  
Schmallenberg Virus ◽  
Rt Pcr ◽  
Bovine Semen ◽  
Bull Semen ◽  
Virus Rna ◽  
Virus Excretion ◽  
Venereal Transmission

Abstract The detection of Schmallenberg virus (SBV) in the breeding bull semen raised the question of the possibility of venereal transmission of SBV which could result in cost-intensive restrictions in the trade of bovine semen. In order to evaluate the presence of SBV RNA in bovine semen, 131 bull semen samples from four locations in Poland collected between 2013 and 2015 were analysed by RT-PCR for viral RNA. SBV RNA was detected in 5.3% of the samples. The study has revealed that application of an appropriate RNA extraction method is crucial to detect virus excretion via semen.

scholarly journals Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

2007 ◽  
Vol 73 (5) ◽  
pp. 1457-1466 ◽  
Author(s):  
Else M. Fykse ◽  
Gunnar Skogan ◽  
William Davies ◽  
Jaran Strand Olsen ◽  
Janet M. Blatny
Keyword(s):  
Nucleic Acid ◽  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Environmental Water ◽  
Nucleic Acid Sequence ◽  
Environmental Water Samples ◽  
Dnase Treatment ◽  
Nasba Assay ◽  
Toxigenic Strains

ABSTRACT A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

scholarly journals A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

2019 ◽  
Author(s):  
Farhad Masoomi-Aladizgeh ◽  
Leila Jabbari ◽  
Reza Khayam Nekouei ◽  
Ali Aalami
Keyword(s):  
Nucleic Acid ◽  
Extraction Method ◽  
Ethylenediaminetetraacetic Acid ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Plant Tissues ◽  
Hexadecyltrimethylammonium Bromide ◽  
Dna And Rna ◽  
Extraction Buffer ◽  
Main Components

Abstract This protocol describes a rapid DNA and RNA extraction method for plant tissues. Hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), tris base, and ethylenediaminetetraacetic acid (EDTA) are the main components of the extraction buffer. In contrast to all previously reported protocols, this extraction method does not require any stock solutions. This isolation buffer is potential of extracting both DNA and RNA simultaneously. Depending on the purpose of the project, the corresponding steps can be slightly altered to obtain either DNA or RNA. The big advantage of this method is to use general laboratory chemicals to make a powerful extraction buffer, resulting in high quality and quantity nucleic acid. Also, CTAB in this buffer is capable of isolating nucleic acid from recalcitrant plants enriched in secondary metabolites. Importantly, this method is recommended for the projects at which isolating nucleic acid in a short time is of crucial importance. This method probably is usable for all plant tissues and takes about an hour.

scholarly journals Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

2021 ◽  
Author(s):  
Santosh Karade ◽  
Pratik Thosani ◽  
Prashant Patil ◽  
Kavita Bala Anand ◽  
Sourav Sen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pilot Study ◽  
Real Time ◽  
Gold Standard ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

scholarly journals Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan

2018 ◽  
Vol 21 (2) ◽  
pp. 106
Author(s):  
Wiwik Endarsih ◽  
Sedyo Hartono ◽  
Sri Sulandari
Keyword(s):  
Nucleic Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Extraction Method ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Viral Pathogen ◽  
Virus Rna ◽  
Tomato Chlorosis Virus ◽  
Commercial Kit

Replicative form (RF) of RNA viruses are dsRNA structured nucleic acid, always found in plants infected by RNA virus. The principle of dsRNA extraction is based on the different affinity of nucleic acids for the cellulose powder and the specific adsorption in 16.6% ethanol buffer. The study aims to develop the simple dsRNA extraction method for the preparation of RT-PCR detection for Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV), and compared with commercial kit. The analysis was performed by quantification of nucleic acid with spectrophotometer, efficiency of method (level of complexity, time, cost per reaction) and sequencing. The RNA concentration with simple methode of dsRNA extraction was lower than kit extraction method but the both methods have same pure RNA result. The PCR and sequencing result showed that viral pathogen of pepper, tobacco, and tomato leaf was CMV, ReMV, and ToCV, respectively with amplicon size at 500, 568, and 360 bp. This method is quite cheap and the RNA quantity is proportional to the commercial kit. The simple method of dsRNA extraction can be proposed for the preparation of RT-PCR detection for CMV, ReMV, ToCV. IntisariReplicative form (RF) virus RNA merupakan asam nukleat berstruktur dsRNA, selalu ditemukan pada tumbuhan terinfeksi oleh virus RNA. Prinsip kerja ekstraksi dsRNA berdasarkan afinitas serbuk selulosa terhadap asam nukleat dan adsorbsi spesifik dsRNA pada konsentrasi etanol 16,6 %. Penelitian ini bertujuan untuk mengembangkan metode ekstraksi dsRNA secara sederhana untuk preparasi deteksi RT-PCR terhadap Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV) dan dibandingkan dengan kit komersil. Data yang dibandingkan adalah kuantitas asam nukleat, analisa efisiensi metode (tingkat kerumitan, waktu, biaya per reaksi) serta sekuensing. Konsentrasi RNA hasil ekstraksi metode dsRNA secara sederhana lebih rendah dibanding dengan metode kit, namun kedua metode menghasilkan RNA yang murni. Berdasarkan hasil PCR dan sekuensing disimpulkan bahwa virus penyebab mosaik daun lada dan tembakau serta klorosis daun tomat berturut-turut adalah CMV, ReMV, dan ToCV dengan ukuran amplikon berturut turut 500, 568 dan 360 pb. Metode ini cukup murah dan kuantitas RNA yang dihasilkan sebanding dengan kit komersil. Ekstraksi RNA menggunakan metode dsRNA secara sederhana dapat dikembangkan untuk preparasi deteksi RT-PCR terhadap CMV, ReMV, ToCV.

scholarly journals Automation of Nucleic Acid Isolation on KingFisher Magnetic Particle Processors

2007 ◽  
Vol 12 (4) ◽  
pp. 195-201 ◽  
Author(s):  
Xingwang Fang ◽  
Roy C. Willis ◽  
Angela Burrell ◽  
Kurt Evans ◽  
Quoc Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Pathogen Detection ◽  
Magnetic Particle ◽  
Rna Isolation ◽  
Viral Rna ◽  
Cell Fractionation ◽  
Rt Pcr ◽  
Total Rna ◽  
Total Rna Isolation

We describe automated nucleic acid (NA) isolation from diverse sample types using MagMAX kits (Ambion, Inc.) on KingFisher Magnetic Particle Processors (Thermo Scientific). The MagMAX-96 Blood RNA Isolation Kit is designed for total RNA isolation from whole blood from several species, without white blood cell fractionation, in about 45 min (including genomic DNA removal). The MagMAX-96 Total RNA Isolation Kit is designed for total RNA isolation from up to 2 × 10 6 cultured cells and up to 10-mg tissue. The isolated total RNA is highly intact and pure, ready to use in downstream applications such as microarray analysis or real-time reverse transcription (RT)-PCR for gene expression profiling or pathogen detection. The MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA and DNA isolation from cell free or nearly cell-free samples such as swabs, serum, and plasma; it takes about 15 min. Total NA of high quality and purity is recovered at >75% efficiency, providing high sensitivity for pathogen detection by real-time RT-PCR. Unlike automated liquid handling systems that move reagents into and out of a single well of a multiwell plate to perform the different steps of an RNA isolation procedure, the KingFisher Magnetic Particle Processors use permanent magnetic rods to collect magnetic beads from solution and release them into another well containing reagent for the subsequent step of the procedure. The effectiveness of bead collection and transfer lead to superior washing and elution efficiency, as well as rapid processing. It is a very effective strategy for automation of magnetic-bead-based NA isolation kits. (JALA 2007; 12:195–201)

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