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.

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 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 Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

2016 ◽  
Vol 54 (7) ◽  
pp. 1766-1773 ◽  
Author(s):  
Monika Simmons ◽  
Todd Myers ◽  
Carolina Guevara ◽  
Donald Jungkind ◽  
Maya Williams ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Sensitivity And Specificity ◽  
Internal Control ◽  
Chikungunya Virus ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Denv Serotypes ◽  
Reverse Transcriptase Pcr ◽  
One Step

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), andin vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

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.

Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus

2006 ◽  
Vol 133 (1) ◽  
pp. 14-19 ◽  
Author(s):  
Enrique Royuela ◽  
Ana Negredo ◽  
Alicia Sánchez-Fauquier
Keyword(s):  
Real Time ◽  
Sensitive Detection ◽  
Rt Pcr ◽  
One Step ◽  
Human Astrovirus ◽  
Pcr Method

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.

Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus

2016 ◽  
Vol 26 (8) ◽  
pp. 1398-1403 ◽  
Author(s):  
Mi-Ju Kim ◽  
Shin-Young Lee ◽  
Hyun-Joong Kim ◽  
Jeong Su Lee ◽  
In Sun Joo ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
One Step ◽  
Pcr Method

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.

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