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.

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 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 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.

scholarly journals First Genome Sequence of Newcastle Disease Virus of Genotype VIIi from Jordan

2018 ◽  
Vol 7 (23) ◽  
Author(s):  
Mustafa Ababneh ◽  
Helena L. Ferreira ◽  
Mohammad Khalifeh ◽  
David L. Suarez ◽  
Claudio L. Afonso
Keyword(s):  
Reverse Transcriptase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Genome Sequence ◽  
Illumina Sequencing ◽  
Newcastle Disease ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Chicken Spleen

Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing.

scholarly journals Detection of Mycobacterium tuberculosis Group Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis

2001 ◽  
Vol 69 (3) ◽  
pp. 1821-1831 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Andrew A. McColm ◽  
Julie A. Bagshaw ◽  
Richard Reece ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mycobacterium Tuberculosis ◽  
Synovial Fluid ◽  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Rt Pcr ◽  
Diverse Range ◽  
Susceptible Host ◽  
Joint Tissue ◽  
Reverse Transcriptase Pcr

ABSTRACT Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model ofM. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosisinfection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

scholarly journals Two variants of quantitative reverse transcriptase PCR used to show differential expression of α-, β- and γ-fibrinogen genes in rat liver lobes

1997 ◽  
Vol 321 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Junlong ZHANG ◽  
Mina DESAI ◽  
Susan E. OZANNE ◽  
Cora DOHERTY ◽  
C. Nicholas HALES ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rat Liver ◽  
Coefficient Of Variation ◽  
Copy Number ◽  
Internal Standard ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Mrna Copy Number ◽  
Sensitive Method

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of α-, β- and γ-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (α-fibrinogen F = 14.64, P = 0.0003; β-fibrinogen F = 3.74, P = 0.04; γ-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

scholarly journals Use of Reverse Transcriptase PCR in Early Diagnosis of Rift Valley Fever

2002 ◽  
Vol 9 (3) ◽  
pp. 713-715 ◽  
Author(s):  
A. A. Sall ◽  
E. A. Macondo ◽  
O. K. Sène ◽  
M. Diagne ◽  
R. Sylla ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Virus Isolation ◽  
Immunoglobulin M ◽  
Antibody Detection ◽  
Rt Pcr ◽  
Valley Fever ◽  
Reverse Transcriptase Pcr ◽  
Sensitive Tool

ABSTRACT Reverse transcriptase PCR (RT-PCR) for diagnosis of Rift Valley fever (RVF) was evaluated by using 293 human and animal sera sampled during an RVF outbreak in Mauritania in 1998. Results of the RT-PCR diagnostic method were compared with those of virus isolation (VI) and detection of immunoglobulin M (IgM) antibodies. Our results showed that RT-PCR is a specific, sensitive tool for RVF diagnosis in the early phase of the disease and that its results do not differ significantly from those obtained by VI. Moreover, the combined results of RT-PCR and IgM antibody detection were in 100% concordance with the results of VI.

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