scholarly journals Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

2002 ◽  
Vol 68 (10) ◽  
pp. 5017-5025 ◽  
Author(s):  
Balbina Nogales ◽  
Kenneth N. Timmis ◽  
David B. Nedwell ◽  
A. Mark Osborn
Keyword(s):  
Reverse Transcription ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Estuarine Sediments ◽  
Rt Pcr ◽  
Sediment Samples ◽  
Reverse Transcription Pcr ◽  
Nitrogen Inputs ◽  
Denitrification Genes

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

scholarly journals An Integrated Diagnostic Approach for Citrus Exocortis Viroid

2021 ◽  
Author(s):  
Chih-Hsu Lin ◽  
Ting-Hsuan Hung ◽  
I Hu ◽  
Ta-Hsin Ku ◽  
Chun-Yi Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Severe Disease ◽  
Blot Hybridization ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Cultivar ◽  
Dot Blot Hybridization ◽  
Reverse Transcription Pcr ◽  
One Step

Abstract BackgroundCitrus exocortis viroid (CEVd) is a circular single-stranded RNA pathogen consists of around 370 nucleotides and leads to a severe disease showing bark scaling symptom on citrus crops, which leads to yield decrease and economic loss. Since the absence of viroid-encoded proteins, methods for CEVd detection mainly counts on bioassays or nucleic acid-base approaches. In order to validate the CEVd disease, here we developed an integrated diagnostic protocol. MethodsCEVd transcripts were inoculated onto two susceptible cultivars of Solanum lycopersicum L., cv. Rutgers and cv. Double-Fortune, seedings. After inoculation, total RNAs of the two tomato cultivars were extracted to detect CEVd infection by dot blot hybridization, one-step reverse transcription PCR (one-step RT-PCR) and real-time reverse transcription PCR (real-time RT-PCR). In addition, the symptom development of both cultivars was recorded weekly. ResultsThe tomato cultivar Rutgers rather than Double-Fortune or others was selected as a suitable CEVd-indicator plant and the bio-index score was established based on epinasty, vein necrosis, leaf size reduction and stunting symptoms. In addition, the isolate of CEVd that collected from citrus field could rapidly and consistently cause the index symptoms on Rutgers. As expected, CEVd could be specifically and sensitively detected in both tomato and citrus plants by dot-blot hybridization and RT-PCR technologies, including one-step RT-PCR and real-time RT-PCR. Furthermore, we found that the levels of CEVd genomic RNA or CEVd derived small RNAs are correlated to symptom severity. ConclusionsIn this study, we developed an integrated detection method for CEVd and revealed potential underlying viroid-host interactions.

scholarly journals Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (10) ◽  
pp. 5996-6004 ◽  
Author(s):  
Jan Vinjé ◽  
Sjon J. G. Oudejans ◽  
Jill R. Stewart ◽  
Mark D. Sobsey ◽  
Sharon C. Long
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Phylogenetic Analyses ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Reverse Line Blot ◽  
Source Tracking ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Male Specific

ABSTRACT In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qβ, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

scholarly journals Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

2001 ◽  
Vol 67 (2) ◽  
pp. 742-749 ◽  
Author(s):  
Kellogg J. Schwab ◽  
Frederick H. Neill ◽  
Françoise Le Guyader ◽  
Mary K. Estes ◽  
Robert L. Atmar
Keyword(s):  
Enzyme Immunoassay ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Product ◽  
Dna Enzyme Immunoassay

ABSTRACT Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

scholarly journals Quantification of Melanoma Cell-specific MART-1 mRNA in Peripheral Blood by a Calibrated Competitive Reverse Transcription-PCR

2000 ◽  
Vol 46 (12) ◽  
pp. 1923-1928 ◽  
Author(s):  
Boe Sandahl Sørensen ◽  
Henrik Schmidt ◽  
Hans von der Maase ◽  
Per Thor Straten ◽  
Ebba Nexø
Keyword(s):  
Malignant Melanoma ◽  
Melanoma Cell ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Pcr Amplification ◽  
Melanoma Cells ◽  
Rt Pcr ◽  
Blood Samples ◽  
Reverse Transcription Pcr

Abstract Background: Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood. Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve. Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 × 10−21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 × 10−21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 × 10−21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 × 10−21 mol MART-1 mRNA/mL), respectively. Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.

scholarly journals Development of Reverse Transcription-PCR (Oligonucleotide Probing) Enzyme-Linked Immunosorbent Assays for Diagnosis and Preliminary Typing of Foot-and-Mouth Disease: a New System Using Simple and Aqueous-Phase Hybridization

2000 ◽  
Vol 38 (12) ◽  
pp. 4604-4613 ◽  
Author(s):  
Soren Alexandersen ◽  
Morag A. Forsyth ◽  
Scott M. Reid ◽  
Graham J. Belsham
Keyword(s):  
Reverse Transcription ◽  
Pcr Amplification ◽  
Foot And Mouth Disease ◽  
Variable Region ◽  
Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mouth Disease Virus ◽  
Foot And Mouth

A reverse transcription-PCR (RT-PCR)–enzyme-linked immunosorbent assay system that detects a relatively conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease virus (FMDV) has been developed. The high specificity of the assay is achieved by including a rapid hybridization step with a biotin-labeled internal oligonucleotide. The assay is highly sensitive, fast, and easy to perform. A similar assay, based on a highly variable region of the FMDV genome and employing a single asymmetric RT-PCR and multiple hybridization oligonucleotides, was developed to demonstrate the method's ability to type FMDV. Based on our theoretical and practical knowledge of the methodology, we predict that similar assays are applicable to diagnosis and strain differentiation in any system amenable to PCR amplification.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

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