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At least 613 records · Page 34Linked to original sources
PubMed2002

Manufacturing DNA microarrays from unpurified PCR products.

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.

Animals↗
PubMed2002

Quality control of translation through the kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.

It is known that each aminoacyl-tRNA synthetase (aaRS) specifically recognizes its cognate tRNAs to ensure the correct translation of the genetic information. However, we had previously demonstrated that mammalian mitochondrial seryl-tRNA synthetase (mt SerRS) can markedly misacylate mitochondrial (mt) tRNA(Gln). To investigate extensively misacylation reactions in mammalian mitochondrion, we purified overall twenty-two mt tRNAs from bovine liver, and determined their misacylation activities using mt SerRS. In addition to tRNA(Gln), tRNA(Ala) and tRNA(Asn) showed weak but significant serylation activities, which raises the possibility that each mammalian mt aaRS can misacylate several non-cognate mt tRNAs in varying degrees, but translational fidelity might be maintained by kinetic discrimination of tRNAs in the network of aaRSs.

Amino Acyl-tRNA Synthetases↗
PubMed2005

Diagnosis of cat scratch disease with Bartonella henselae infection in formalin-fixed paraffin-embedded tissues by two different PCR assays.

Cat scratch disease (CSD) is commonly caused by Bartonella henselae infection. Clinical history and histologic findings are often insufficient to establish a definitive diagnosis of CSD. We retrospectively studied formalin-fixed, paraffin-embedded (FFPE) lymph nodes from 35 patients with histologically suspected CSD by 2 different PCR assays and immunohistochemistry (IHC). The first primer pair amplified a 163-bp fragment of the 16S rRNA gene in 19 of the 35 cases (54%). The second primer pair amplified a 191-bp fragment of the henselae citrate synthase (gltA) gene in 17 of the 35 cases (49%). IHC identified the organisms in 8 of 33 cases (24%). Fresh cultures of various Bartonella species showed a specific PCR product with an analytical sensitivity of 0.5 to 5 pg bacterial DNA. Bartonella species were identified by the unique size of the amplified PCR product. Twenty-two lymph nodes without morphologic evidence or a history of CSD were negative by PCR and immunostaining. Tissues from a patient with Legionella pneumophila were also negative by PCR and immunostaining for CSD supporting the specificity of the PCR reaction. The specific PCR products of the B. henselae were confirmed by sequencing. Human beta-actin for each case was amplified to check the integrity of the DNA. Our data indicate that detection of Bartonella DNA by PCR is useful to confirm the diagnosis of CSD.

Actins↗
PubMed2001

Nocardia ignorata sp. nov.

An isolate that was received during a mycobacterial quality control test and which was thought to be a Mycobacterium species was subjected to a polyphasic taxonomic study after mycolic acid analysis showed that it possessed mycolates characteristic of the nocardiae. Further chemotaxonomic and 16S rRNA gene sequence analyses of this isolate demonstrated that it belongs to the genus Nocardia. 16S rRNA gene sequence data, DNA-DNA hybridization studies and the biochemical characteristics of the isolate indicate that it belongs to a novel species that differs from previously described members of the genus Nocardia. The name Nocardia ignorata sp. nov. is proposed for this isolate with the type strain IMMIB R-1434T (= DSM 44496T = NRRL B-24141T).

DNA, Ribosomal↗
PubMed2003

Characterization of four orthologs of stringent starvation protein A.

Orthologous proteins can be beneficial for X-ray crystallographic studies when a protein from an organism of choice fails to crystallize or the crystals are not suitable for structure determination. Their amino-acid sequences should be similar enough that they will share the same fold, but different enough so that they may crystallize under alternative conditions and diffract to higher resolution. This multi-species approach was employed to obtain diffraction-quality crystals of the RNA polymerase (RNAP) associated stringent starvation protein A (SspA). Although Escherichia coli SspA could be crystallized, the crystals failed to diffract well enough for structure determination. Therefore, SspA proteins from Yersinia pestis, Vibrio cholerae and Pseudomonas aeruginosa were cloned, expressed, purified and subjected to crystallization trials. The V. cholerae SspA protein failed to crystallize under any conditions tested and the P. aeruginosa SspA protein did not form crystals suitable for data collection. On the other hand, Y. pestis SspA crystallized readily and the crystals diffracted to 2.0 A.

Amino Acid Sequence↗
PubMed2006

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment.

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.

Bacteriological Techniques↗
PubMed2005

Polymerase chain reaction inhibition assay documenting the amotosalen-based photochemical pathogen inactivation process of platelet concentrates.

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system. STUDY DESIGN AND METHODS: To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci. RESULTS: Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT. CONCLUSION: Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.

Adenine↗
PubMed2006

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene.

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.

Animals↗
PubMed1999

Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results.

A polymerase chain reaction assay (PCR) for the diagnosis of Helicobacter pylori in human gastric biopsies was developed. To prevent false-negative results while performing PCR on human tissues, an internal control is necessary. Primer set ACT1-ACT2 which specifically amplifies a 542-bp fragment of the 16S rRNA gene of H. pylori was used. dUTP and hot-start were used to prevent false-positives from carryover of previous products and avoid non-specific extension products. A competitive internal control DNA fragment was constructed to detect the presence of inhibitors. Biopsies from 101 unselected patients with gastric symptoms were tested. PCR results were compared with results from microscopy of histological sections and conventional culturing for H. pylori. Forty-two percent of the biopsies were found to contain compounds inhibiting the PCR. The addition of the internal control assures the performance of the PCR assay and is an important quality control parameter.

Adolescent↗
PubMed2005

Addition of poly(A) and heteropolymeric 3' ends in Bacillus subtilis wild-type and polynucleotide phosphorylase-deficient strains.

Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli, poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3' tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3'-tail addition in Bacillus subtilis, which lacks an identifiable PAP I homologue, was studied. Sizing of poly(A) sequences revealed a similar pattern in wild-type and PNPase-deficient strains. Sequencing of 152 cloned cDNAs, representing 3'-end sequences of nontranslated and translated RNAs, revealed modified ends mostly on incomplete transcripts, which are likely to be decay intermediates. The 3'-end additions consisted of either short poly(A) sequences or longer heteropolymeric ends with a mean size of about 40 nucleotides. Interestingly, multiple independent clones exhibited complex heteropolymeric ends of very similar but not identical nucleotide sequences. Similar polyadenylated and heteropolymeric ends were observed at 3' ends of RNA isolated from wild-type and pnpA mutant strains. These data demonstrated that, unlike the case of some other bacterial species and chloroplasts, PNPase of Bacillus subtilis is not the major enzyme responsible for the addition of nucleotides to RNA 3' ends.

Bacillus subtilis↗
PubMed2001

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species.

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

Databases, Nucleic Acid↗
PubMed2003

Human herpesvirus 8 load in matched serum and plasma samples of patients with AIDS-associated Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 x 10(6) copies of HHV-8 DNA (r(2) > 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.

AIDS-Related Opportunistic Infections↗
PubMed2005

Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools.

Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools.

Adenoviridae↗