Universal Eubacterial Primers Research Articles

Colonic mucosal bacterial diversity of de novo extensive paediatric ulcerative colitis by next-generation sequencing

IntroductionDysbiosis may contribute to inflammatory bowel disease (IBD) pathogenesis along with a reduced bacterial diversity. Limited bacterial diversity studies have been performed at the onset of disease in adults but...

MICROBIOLOGICAL QUALITY OF THE GHANAIAN VOLTA CLAM (GALATEA PARADOXA)

ABSTRACTMicrobiological quality of the Ghanaian Volta clam (Galatea paradoxa) was assessed by fecal coliform and total aerobic bacteria counts. Polymerase chain reaction (PCR) with universal eubacterial 16S and 23S rDNA primers was also used to show the presence of bacteria in samples of the gut fluid and mantle tissues. The clams were found to be laden with potential pathogenic bacteria identified by conventional methods. Twenty bacteria species were isolated. They were Pseudomonas aeruginosa, Klebsiella ornithinolytica, Flavobacterium meningosepticum, Enterobacter aerogenes, E. agglomerans, E. cloacae, Aeromonas sobria, Acinetobacter sp, Vibrio cholerae, Micrococcus radiodurans, Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, Citrobacter freundii, Chromobacterium violaceum, Morganella morganii, Proteus mirabilis, Salmonella sp., Serratia marcescens and Yersinia intermedia. With PCR, bacterial DNA fragments were detected in 16.7% (2/12) of clam gut and 83.3% (10/12) of mantle tissues. Bacteria load in the clam mantle tissue was higher than in the gut.PRACTICAL APPLICATIONSThe Volta clam is of high commercial interest in the Volta estuary. Information on the presence of potential pathogenic bacteria in this shellfish is thus very important to the food industry, especially in the shellfish‐eating community, where the shellfish is eaten in the semi‐processed ready‐to‐eat state. Data from this article will assist to ensure food safety.

Endocarditis Caused by Actinobaculum schaalii, Austria

Endocarditis Caused by Actinobaculum schaalii, Austria

Characterization and evolution of two bacteriome‐inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini)

Like other plant sap-sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with 'Candidatus Sulcia muelleri' was detected in PCR assays using primers specific for the Bacteroidetes. Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola, fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that 'Ca. Sulcia muelleri' and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. 'Candidatus Purcelliella pentastirinorum' was proposed as the novel gammaproteobacterial symbiont.

Analysis of the Structure of the Bacterial Community in the Livestock Manure-based Composting Process

: We investigated the structure of bacterial communities present in livestock manure-based composting processes and evaluated the bacterial succession during the composting processes. Compost samples were derived separately from swine manure, dairy manure and sewage sludge. The structure of the bacterial community was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) using universal eubacterial primers. The genus Bacillus and related genera were mainly detected following the thermophilic composting phase of swine and dairy manure composts, and the members of the phylum Bacteroidetes were mainly detected in the cattle manure waste-based and sewage sludge compost. We recovered and sequenced limited number of the bands; however, the PCR-DGGE analysis showed that predominant diversities during the composting processes were markedly changed. Although PCR-DGGE analysis revealed the presence of different phyla in the early stages of composting, the members of the phylum Firmicutes and Bacteroidetes were observed to be one of the predominant phyla after the thermophilic phase.

Detection and identification of unknown streptococcal populations in clinical samples

Background and aim : Streptococci are a heterogeneous group that includes more than 30 different species. Most of the species are usual components of the human flora and some are important human pathogens. To date, molecular tools have not been developed for studying the diversity of the genus Streptococcus in clinical samples. We have developed a PCRbased analytical tool to investigate the presence of new yet uncultured pathogenic streptococci in clinical samples. Methods : A 16S rRNA gene-targeted oligonucleotide (1043F) covering all species of the genus Streptococcus has been designed and used in combination with the universal eubacterial primer, 1492R, to produce approximately 450 bp PCR fragments that enclose three (V7, V8 and V9) hypervariable regions. PCR products are suitable for separation through denaturing gradient gel electrophoresis (DGGE). The inclusivity of the PCR assay was verifi ed on 16 different Streptococcus species representing all current taxonomic groups. The exclusivity of the primers was tested on 29 other bacterial species representing all major phylogenetic lineages. Results : Positive PCR results were obtained for all Streptococcus species and Enterococcus faecalis, whereas PCR was negative for the other specimens. The amplifi cation limit was 170 genome equivalents per reaction (95% probability). The oligonucleotide set was tested on colon biopsies and bronchoalveolar lavage samples. Thus, from the 33 sequences retrieved, 16 (48.5%) shared identity to cultured streptococci, whereas 11 (33.3%) of the sequences belonged to uncultured streptococci and three (9%) to other genera. Three sequences (9.7%) corresponded to new Streptococcus phylotypes (< 95% identified to previously known sequences). Conclusion : The use of this new primer set in combination with DGGE has proven to be a suitable method for studying the specific composition of streptococcal populations, particularly those from infected tissues. Moreover, this method can be of use in the identifi cation of new Streptococcus species that are potentially involved in polymicrobial infections. Key words: DGGE, microbial ecology, polymicrobial infections

Influence of 16S rDNA primer sequence mismatches on the spectrum of bacterial genera detected in prostate tissue by universal eubacterial PCR

Propionibacterium sp. and Staphylococcus spp. are the most frequent bacteria cultured from prostatectomy specimens but are seldom detected by universal eubacterial PCR. We obtained from GenBank representative 16S rRNA gene sequences from Propionibacterium sp., Staphylococcus spp. and from 34 bacterial genera that were recently detected in prostate tissues using universal eubacterial PCR. We compared these 16S rDNA sequences with the universal eubacterial 16S PCR primer sets chosen for detection of bacterial DNA in prostate tissues. We show that failure to detect DNA from Propionibacterium sp. and Staphylococcus spp. in prostate tissues is strongly associated with the presence of mismatches near the 3' termini of the 16S rDNA primer sets used. The choice of 16S PCR primers may play an important role in determining the spectrum of bacterial genera detected in prostate tissue by universal eubacterial PCR.

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

Seasonal Hypoxia and the Genetic Diversity of Prokaryote Populations in the Central Basin Hypolimnion of Lake Erie: Evidence for Abundant Cyanobacteria and Photosynthesis

The reoccurring region of seasonal hypoxia in the central basin of Lake Erie (“ the dead zone”) has been of significant interest to researchers over the past several years. Surprisingly however, no efforts to characterize the endemic microbial community, responsible for the consumption of oxygen in this system, have been published. To understand how the microbial community may be interacting with this event, we have begun to characterize microbial members by using molecular tools. Phycoerythrinrich cyanobacteria appear abundant and active in a narrow region (∼ 1.5 m) below the thermocline during hypoxic conditions, reaching abundances of greater than 10 5 mL −1 and being the primary agent releasing 1.5 mg O 2 L −1 above the daytime demands in this region. Sequencing of 16S rDNA amplicons, generated with universal eubacterial primer sets, from the Lake Erie's hypolimnion during seasonal oxygen depletion demonstrated that cyanobacteria, most closely related to phycoerythrin-rich Synechococcus spp., dominate during rapid drawdown of oxygen (0.083 mg L −1 d −1 in 2004) in this region. Analyses of another conserved marker of phylogeny (RuBisCO) has been used to confirm the presence of these cell types. Numerous distinct taxa of heterotrophic bacteria are also represented in the 16S library. The results of this study suggest that novel groups of cyanobacteria may persist within the Lake Erie dead zone during hypoxic conditions and, along with the heterotrophic community, strongly influence system geochemistry.

Detection of Actinobacillus actinomycetemcomitans But Not Bacteria of the Red Complex in Aortic Aneurysms by Multiplex Polymerase Chain Reaction

Aortic aneurysms affect an increasing number of elderly patients and cause considerable morbidity and mortality. The understanding of the mechanisms involved in the pathogenesis of aortic aneurysms is unclear and little is known about the role of microorganisms in the development of the condition. The aim of the present study was to examine aortic aneurysm samples for the presence of four putative periodontal pathogens: Actinobacillus actinomycetemcomitans, Treponema denticola, Tannerella forsythensis, and Porphyromonas gingivalis. Fifty-six samples from the aneurysm wall were obtained from patients undergoing aneurysm repair. DNA was extracted from tissue by conventional methods. Universal eubacterial primers for general detection of bacteria and species specific primers for detection of the periodontal pathogens were used to amplify part of the 16S rRNA gene by polymerase chain reaction (PCR). Bacterial DNA was detected in 50 of the 56 aneurysm samples (89.2%). A. actinomycetemcomitans was found in four samples (7.1%). None of the samples was positive for T. denticola, T. forsythensis, or P. gingivalis. Bacteria are commonly present in aortic aneurysms and may play a role in the development of the condition. Periodontal pathogens are also present.

Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the gamma- and delta-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.

Molecular screening of unculturable bacteria present in men with reproductive failure.

Objective: To determine the presence of unculturable bacteria using polymerase chain reaction (PCR) in infertile men with pyosperrmia. Design: Perspective clinical study. Setting: The study took place at the Department of Reproductive Medicine, Owaisi Hospital and Research Center; In vitro Fertilization Unit, Mahavir Hospital and Research Center; Center for Cellular and Molecular Biology; and Bharat Biotech Foundation, Hyderabad, India. Patients: A total of 68 infertile men and 15 donors, all with no symptoms of genito-urinary tract infections and sterile semen cultures were included in the study. Interventions: None. Main outcome measures: Screening bacteria using routine bacterial cultures and PCR based screening with universal eubacterial primers. Results: The statistical analysis of all the semen parameters in asthenazoospermic, azoospermic, ceyptazoospermic, severe oligospermic and mild oligospermic patients were found to be significant compared with the controls. All the groups were found to be significant compared with the controls (P<0.05) except for volume and pus cells in the cryptozoospermia group. The Student's t-test also was significant for the seminal parameters before and after treatment of 68 selected individuals with pyospermia and sterile cultures. A total of 44.11% (30/68) samples were collected from the negative culture of pyospermic infertile men have shown the presence of bacteria on amplification using PCR with universal eubacterial primers. The DNA was purified and sequenced. The sequences were checked for homology using DNASTAR and Ribosomal DataBase Project II. A total of 90% of the samples have shown the nearest evolutionary relation to Pantoea P102 (AF394539) and 10% of samples have shown close relation with Burkholderia cepacia (AF042161). Conclusion: The routine bacteriological cultures were unable to detect certain bacterial species particularly with members of enterobacteriaceae family (Pantoea species). Polymerase chain reaction, when used for screening bacteria, can detect the unculturable form of bacteria in infertile men. No amplification for bacterial DNA was obtained in control samples (fertile men with sterile semen cultures.) (Reprod Med Biol 2004; 3: 77- 84).

Assessment of equine fecal contamination: the search for alternative bacterial source-tracking targets

16S rDNA clone libraries were evaluated for detection of fecal source-identifying bacteria from a collapsed equine manure pile. Libraries were constructed using universal eubacterial primers and Bacteroides- Prevotella group-specific primers. Eubacterial sequences indicated that upstream and downstream water samples were predominantly β- and γ-Proteobacteria (35 and 19%, respectively), while the manure library consisted predominantly of Firmicutes (31%) and previously unidentified sequences (60%). Manure-specific eubacterial sequences were not detectable beyond 5 m downstream of the pile, suggesting either poor survival or high dilution rates. In contrast, Bacteroides and Prevotella sp. sequences were detected both in manure and downstream using group-specific primers. Novel sequences from Bacteroides and Prevotella analysis produced an equine-specific phylogenetic cluster as compared to previous data sets obtained for human and bovine samples. While these results suggest that some anaerobic fecal bacteria might be potential identifiers for use in source-tracking applications, a comprehensive examination of environmental sequences within these species should be performed before methods targeting these bacterial groups are applied to watersheds for development of microbial source-tracking protocols.

Molecular evidence of Coxiella-like microorganism harbored by Haemaphysalis concinnae ticks in the Russian Far East.

Twenty female imago of Haemaphysalis concinnae ticks collected from wild vegetation in May of 2001 in the Obor region near Khabarovsk in the Russian Far East were studied. DNA was extracted with commercially available kits following manufacturer's instructions. We used broad range primers SFG3 and SFG6 proposed for amplification of 16S ribosomal RNA gene's portion of spotted fever group Rickettsia. Initial amplification resulted in positive results in all 20 ticks. Direct sequencing of five randomly chosen amplicons showed that obtained sequences belong to a new species. Closest homology was found in Coxiella burnetii 16S rDNA. Using the obtained sequence as a basis, we designed internal primers highly specific for that sequence. Then we confirmed our results by nested PCR amplification with newly designed primers Cox1 and Cox2-all 20 samples also were positive. Having combined these primers with universal eubacterial primers fD1 and rP2 we retrieved a 1,043 bp portion of the 16S rRNA gene. Consecutive homology search demonstrated that the closest sequence similarity belongs to new Coxiella sp. recently found in Rhipicephalus sanguineus ticks (95.2% of homology) and Coxiella burnetii (94% of homology). So, our results suggest that a novel Coxiella-like microorganism, provisionally called here "Cenerentola," is harbored by Haemaphysalis concinnae ticks in the Russian Far East.

Application of temperature gradient gel electrophoresis to the characterization of a nitrifying bioaugmentation product

The microbial population of a nitrifying bioaugmentation product (NBP) has been examined using a combination of conventional bacteriological methods and modern molecular techniques. Variable region 3 (V3) of the 16S rRNA genes of the bacteria in NBP was amplified via the polymerase chain reaction (PCR) with universal eubacterial primers and analyzed via temperature gradient gel electrophoresis (TGGE). Two of the predominant PCR products in NBP were purified from the TGGE gel matrix, reamplified via PCR and sequenced. Two nitrifying strains (NS500-9 and MPN2) that had been isolated from the NBP mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR-amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the NBP, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter species, respectively, in the database. Samples from a laboratory-scale bioreactor, bioaugmented with NBP, were used in an attempt to correlate an increase in activity with a detectable shift in the population of NS500-9 and MPN2 via TGGE. No detectable shift in population was observed in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when NBP was present in activated sludge. In biomass spiking experiments as well as in genomic DNA spiking experiments, it was found that NBP must be present at a level of at least 5% of the total population in order to be detected, whereas bioaugmentation at 1% of the total population was enough to yield significant improvements in nitrification efficiency. This study demonstrates how community profiling of an undefined microbial population via TGGE can be used to examine the biological importance of organisms isolated from the same mixed population.

Biodegradation of an alicyclic hydrocarbon by a sulfate-reducing enrichment from a gas condensate-contaminated aquifer.

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.

Rapid detection of columnaris disease in channel catfish ( Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.

Application of temperature gradient gel electrophoresis to the characterization of a nitrifying bioaugmentation product

The microbial population of a nitrifying bioaugmentation product (NBP) has been examined using a combination of conventional bacteriological methods and modern molecular techniques. Variable region 3 (V3) of the 16S rRNA genes of the bacteria in NBP was amplified via the polymerase chain reaction (PCR) with universal eubacterial primers and analyzed via temperature gradient gel electrophoresis (TGGE). Two of the predominant PCR products in NBP were purified from the TGGE gel matrix, reamplified via PCR and sequenced. Two nitrifying strains (NS500-9 and MPN2) that had been isolated from the NBP mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR-amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the NBP, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter species, respectively, in the database. Samples from a laboratory-scale bioreactor, bioaugmented with NBP, were used in an attempt to correlate an increase in activity with a detectable shift in the population of NS500-9 and MPN2 via TGGE. No detectable shift in population was observed in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when NBP was present in activated sludge. In biomass spiking experiments as well as in genomic DNA spiking experiments, it was found that NBP must be present at a level of at least 5% of the total population in order to be detected, whereas bioaugmentation at 1% of the total population was enough to yield significant improvements in nitrification efficiency. This study demonstrates how community profiling of an undefined microbial population via TGGE can be used to examine the biological importance of organisms isolated from the same mixed population.

Optimization of Nested Polymerase Chain Reaction Assays for Identification ofAeromonas salmonicida,Yersinia ruckeri, andFlavobacterium psychrophilum

Nested polymerase chain reaction (PCR) assays were developed using first-round primers complementary to highly conserved regions within the bacterial 16S ribosomal RNA (rRNA) gene (universal eubacterial primers) and second-round primers specific for sequences within the 16S rRNA genes of Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum. Following optimization of the MgCl2 concentration and primer annealing temperature, PCR employing the universal eubacterial primers was used to amplify a 1,500-base-pair (bp) product visible in agarose gels stained with ethidium bromide. The calculated detection limit of this single-round assay was less than 1.4 × 104 colony-forming units (CFU) per reaction for all bacterial species tested. Single-round PCR using primer sets specific for A. salmonicida, Y. ruckeri, and F. psychrophilum amplified bands of 271, 575, and 1,100 bp, respectively, with detection limits of less than 1.4 × 104, 1.4 × 105, and 1.4 × 105 CFU per reaction. Using the universal eubacterial primers in the first round and the species-specific primer sets in the second round of nested PCR assays improved the detection ability by approximately four orders of magnitude to fewer than 14 CFU per sample for each of the three bacterial species. Such nested assays could be adapted to a wide variety of bacterial fish pathogens for which 16S sequences are available.

Reliability of nested polymerase chain reaction in the diagnosis of bacterial endophthalmitis

PURPOSE: To test the utility of DNA Taq polymerase (Taq) treated with 8-methoxypsoralen with ultraviolet irradiation and Taq treated with restriction endonuclease in a nested polymerase chain reaction test for the diagnosis of bacterial endophthalmitis. METHODS: Prospective, comparative study. Vitreous biopsy fluid from 32 cases of clinically diagnosed bacterial endophthalmitis and 10 noninfective controls were cultured for aerobic/anaerobic bacteria and tested by nested polymerase chain reaction using two sets of universal eubacterial primers in triplicate with untreated Taq, Taq treated with 8-methoxypsoralen with ultraviolet irradiation, and Taq treated with Sau 3A1. RESULTS: Using untreated Taq, false-positive results were obtained in nested polymerase chain reaction with all 10 control samples, which were not seen with the other two methods of nested polymerase chain reaction. However, the senstivity of nested polymerase chain reaction using Sau 3A1 was the same sensitivity as the conventional culture (34.4%), whereas the sensitivity of the nested polymerase chain reaction using 8-methoxypsoralen was 46.9% higher than in the conventional culture. CONCLUSION: To eliminate the problem of false positives in bacterial nested polymerase chain reaction, we recommend the routine utilization of Taq treated with 8- methoxypsoralen and ultraviolet irradiation.