scholarly journals Halovirus HF2 Intergenic Repeat Sequences Carry Promoters

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2388
Author(s):  
Brendan Russ ◽  
Friedhelm Pfeiffer ◽  
Mike Dyall-Smith
Keyword(s):  
Comparative Genomics ◽  
Reverse Transcriptase ◽  
Experimental Evidence ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Regulatory Role ◽  
Rt Pcr ◽  
Repeat Sequences ◽  
Pcr Method ◽  
Coding Potential

Halovirus HF2 was the first member of the Haloferacalesvirus genus to have its genome fully sequenced, which revealed two classes of intergenic repeat (IR) sequences: class I repeats of 58 bp in length, and class II repeats of 29 bp in length. Both classes of repeat contain AT-rich motifs that were conjectured to represent promoters. In the present study, nine IRs were cloned upstream of the bgaH reporter gene, and all displayed promoter activity, providing experimental evidence for the previous conjecture. Comparative genomics showed that IR sequences and their relative genomic positions were strongly conserved among other members of the same virus genus. The transcription of HF2 was also examined by the reverse-transcriptase-PCR (RT-PCR) method, which demonstrated very long transcripts were produced that together covered most of the genome, and from both strands. The presence of long counter transcripts suggests a regulatory role or possibly unrecognized coding potential.

A Reverse Transcriptase PCR Technique for the Detection and Viability Assessment of Kluyveromyces marxianus in Yoghurt

2006 ◽  
Vol 69 (9) ◽  
pp. 2210-2216 ◽  
Author(s):  
MARÍA BELÉN MAYORAL ◽  
ROSARIO MARTÍN ◽  
PABLO E. HERNÁNDEZ ◽  
ISABEL GONZÁLEZ ◽  
TERESA GARCÍA
Keyword(s):  
Reverse Transcriptase ◽  
Kluyveromyces Marxianus ◽  
18S Rrna ◽  
Yeast Cells ◽  
Rt Pcr ◽  
Viability Assessment ◽  
Reverse Transcriptase Pcr ◽  
Low Concentrations ◽  
Pcr Method ◽  
High Level

A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 102 CFU ml−1 in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.

scholarly journals Validation of a Methodology for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva by Real-Time Reverse Transcriptase-PCR

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniel F. Escobar ◽  
Pablo Díaz ◽  
Diego Díaz-Dinamarca ◽  
Rodrigo Puentes ◽  
Pedro Alarcón ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Health Authorities ◽  
Specific Alternative ◽  
Pcr Method ◽  
Sensitivity Specificity ◽  
Study Participants

In January 2021, the Chilean city of Concepción experienced a second wave of coronavirus 2019 (COVID-19) while in early April 2021, the entire country faced the same situation. This outbreak generated the need to modify and validate a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva, thereby expanding the capacity and versatility of testing for COVID-19. This study was conducted in February 2021 in the Chilean city of Concepción during which time, the town was under total quarantine. The study participants were mostly symptomatic (87.4%), not hospitalized, and attended care centers because of their health status rather than being asked by the researchers. People coming to the health center in Concepción to be tested for COVID-19 (via reverse transcriptase polymerase chain reaction [RT-PCR]) from a specimen of nasopharyngeal swab (NPS) were then invited to participate in this study. A total of 131 participants agreed to sign an informed consent and to provide saliva and NPS specimens to validate a method in terms of sensitivity, specificity, and statistical analysis of the cycle threshold (Ct) values from the RT-PCR. Calculations pertaining to the 127 participants who were ultimately included in the analysis showed sensitivity and specificity at 94.34% (95% CI: 84.34–98.82%) and 98.65% (95% CI: 92.70–99.97%), respectively. The saliva specimen showed a performance comparable to NPS as demonstrated by the diagnostic parameters. This RT-PCR method from the saliva specimen is a highly sensitive and specific alternative compared to the reference methodology, which uses the NPS specimen. This modified and validated method is intended for use in the in vitro diagnosis of SARS-CoV-2, which provides health authorities in Chile and local laboratories with a real testing alternative to RT-PCR from NPS.

Rapid and Direct Quantitative RT-PCR Method To Measure Promoter Activity

2008 ◽  
Vol 22 (5) ◽  
pp. 1461-1463 ◽  
Author(s):  
Lei Yu ◽  
Frederick E. Domann
Keyword(s):  
Promoter Activity ◽  
Rt Pcr ◽  
Pcr Method

scholarly journals One-Step Reverse Transcriptase PCR Method for Detection of Borrelia burgdorferi mRNA in Mouse Lyme Arthritis Tissue Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 2037-2039 ◽  
Author(s):  
F. X. Limbach ◽  
B. Jaulhac ◽  
Y. Piémont ◽  
J. L. Kuntz ◽  
H. Monteil ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Reverse Transcriptase ◽  
Simple Procedure ◽  
Lyme Arthritis ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Reverse Transcriptase Pcr ◽  
C3h Mice ◽  
One Step ◽  
Pcr Method

A one-step reverse transcriptase PCR (RT-PCR) method for detection of Borrelia burgdorferi mRNA in infected C3H mice is described. This simple procedure, less prone to nucleic acid cross-contamination than the standard method, was found to be 10-fold more sensitive than a classical two-step RT-PCR assay. By using one-step RT-PCR, flagellin mRNAs were detected in synovial and heart tissues from all seven infected mice tested.

scholarly journals Antimicrobial Susceptibility Testing ofChlamydia trachomatis Using a Reverse Transcriptase PCR-Based Method

1999 ◽  
Vol 43 (9) ◽  
pp. 2311-2313 ◽  
Author(s):  
N. A. Cross ◽  
D. J. Kellock ◽  
G. R. Kinghorn ◽  
M. Taraktchoglou ◽  
E. Bataki ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Conventional Method ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Conventional Technique ◽  
Antimicrobial Susceptibility Testing ◽  
Rt Pcr ◽  
Ofchlamydia Trachomatis ◽  
Reverse Transcriptase Pcr ◽  
Pcr Method

ABSTRACT The conventional method for antimicrobial susceptibility testing ofChlamydia trachomatis is subjective and potentially misleading. We have developed a reverse transcriptase PCR (RT-PCR)-based method which is more sensitive and less subjective than the conventional method. Using 16 strains of C. trachomatisin triplicate assays, we found the RT-PCR method consistently more sensitive than the conventional technique for all eight antimicrobials tested, with resultant MICs determined by RT-PCR ranging from 1.6-fold higher (erythromycin) to ≥195-fold higher (amoxicillin).

scholarly journals Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR

2000 ◽  
Vol 66 (7) ◽  
pp. 3119-3124 ◽  
Author(s):  
Sabino Zani ◽  
Mark T. Mellon ◽  
Jackie L. Collier ◽  
Jonathan P. Zehr
Keyword(s):  
New York ◽  
Reverse Transcriptase ◽  
Filamentous Cyanobacteria ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Individual Sequences ◽  
Nitrogenase Genes ◽  
Pcr Method ◽  
Diverse Groups ◽  
Lake George

ABSTRACT A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-μm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the α- and γ-subdivisions of the division Proteobacteria (α- and γ-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, α-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

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