Comparison of storage methods for reverse-transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples

2004 ◽  
Vol 116 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Christopher A Whittier ◽  
William Horne ◽  
Barrett Slenning ◽  
Michael Loomis ◽  
Michael K Stoskopf
2000 ◽  
Vol 68 (10) ◽  
pp. 6012-6026 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Michael Hurle ◽  
Anthony Wong ◽  
Scott Wilkie ◽  
...  

ABSTRACT Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.


2014 ◽  
Vol 77 (11) ◽  
pp. 1953-1959 ◽  
Author(s):  
CAROLINA QUIROZ-SANTIAGO ◽  
CARLOS VÁZQUEZ-SALINAS ◽  
IVAN NATIVIDAD-BONIFACIO ◽  
BLANCA LILIA BARRÓN-ROMERO ◽  
ELSA IRMA QUIÑONES-RAMÍREZ

Rotaviruses are the principal cause of dehydration caused by diarrhea in children younger than 2 years of age. Although these viral infections have mainly been associated with ingestion of fecally contaminated food and water, few studies have addressed the presence of the virus in food that is consumed raw or slightly cooked. In this work, 30 oyster samples and 33 vegetable samples were examined for the presence of rotavirus genotypes to evaluate their potential to produce gastrointestinal infections. The rotaviruses were identified by reverse transcriptase PCR amplification of the VP7 gene. G and P genotyping was also performed by reverse transcriptase PCR, with a detection sensitivity of up to 15 PFU/ml. Rotaviruses were found in 17 (26.9%) of 63 samples (10 oysters and 7 vegetables). The G2 genotype was found in 11 (64.7%) of 17 of the rotavirus strains, and 16 (94.1%) of 17 had the P[4] genotype. The combined genotypes found most frequently were G2P[4] (10 [58.82%] of 17), GNTP[4] (6 [35.29%] of 17), and G2P[NT] (1 [5.8%] of 17).


2005 ◽  
Vol 71 (3) ◽  
pp. 1267-1275 ◽  
Author(s):  
Lina M. Botero ◽  
Seth D'Imperio ◽  
Mark Burr ◽  
Timothy R. McDermott ◽  
Mark Young ◽  
...  

ABSTRACT We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the α-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.


1998 ◽  
Vol 36 (6) ◽  
pp. 1793-1794 ◽  
Author(s):  
James A. Higgins ◽  
Suzana Radulovic ◽  
Bruce H. Noden ◽  
Jill M. Troyer ◽  
Abdu F. Azad

We developed a reverse transcriptase PCR assay to detect expression of 120- and 17-kDa antigen genes in Rickettsia typhi. Infected Vero cell and flea RNAs were reverse transcribed by using random hexamers. The cDNA was amplified by using high concentrations of primer and template in an inexpensive, nonradioactive assay.


2000 ◽  
Vol 68 (12) ◽  
pp. 7166-7171 ◽  
Author(s):  
Heng H. Khun ◽  
Vinay Deved ◽  
Howard Wong ◽  
B. Craig Lee

ABSTRACT The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with anfbpC-sequence-specific oligonucleotide indicates thatfbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.


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