Published Polymerase Chain Reaction Research Articles

A preliminary study of Salmonella spp., Listeria monocytogenes, Escherichia coli O157, Enterococcus faecalis and Clostridium spp. in Irish cattle

Although Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. present a significant food safety and/or spoilage issue for the beef sector, there are limited data on their prevalence in Irish cattle. The objectives of this preliminary study were to investigate the distribution (percentage of farms positive) of Salmonella spp., E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. and the overall prevalence (%) of these bacteria in cattle on a small cohort of Irish beef farms. A total of 121 fresh bovine faecal samples were obtained on 10 randomly selected beef farms in the Northeast of Ireland and tested for the target pathogens using standard culture-based methods. Presumptive positives were confirmed using previously published polymerase chain reaction (PCR) methods. Salmonella were not detected in any of the samples. E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. were present on 50%, 40%, 100% and 100% of farms, respectively, with overall (all farms) prevalence rates in cattle of 9%, 8.2%, 61.9% and 87.6%, respectively. This study suggests that E. coli O157 may be more prevalent than previously thought and L. monocytogenes, E. faecalis and Clostridium spp. are widespread in Irish beef animals.

Molecular and serological characterization of Riemerella isolates associated with poultry in Australia

ABSTRACT A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars.

Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis

BackgroundThe characterization of Leishmania species is important for clinical management of the diseases and the epidemiological control of the parasite distribution. Most of the published polymerase chain reaction (PCR) amplification methods lack the ability to identify all species complexes, have low performance and usually need post-PCR procedures. There is a need for improving the diagnosis of CL by development of an accurate affordable PCR method to identify all Leishmania species in clinical specimens.MethodsWe designed an optimized a PCR amplifying the internal transcribed spacer 2 sequence of the ribosomal RNA gene (ITS2) aligned from different strains of CL-causing Leishmania species in the Old World. The performance of the method was evaluated on lesion samples from several CL suspected patients and the limit of detection (LOD) was determined on DNA of promastigotes from reference strains.ResultsThe universal PCR enabled simultaneous discrimination of L. major, L. tropica and L. infantum using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium culture alone detected 77.27% (17/22) and 72.73% (16/22) of the positive CL samples, respectively. The PCR assay showed 100% sensitivity (22/22) (95% CI: 84.56–100%) and 100% specificity (3/3) (95% CI: 29.24–100%) for species identification on isolates from lesion scraping/exudate and 100% sensitivity (13/13) (95% CI: 75.29–100%) and 100% specificity (11/11) (95% CI: 71.51–100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01–0.1 pg of Leishmania DNA from cultured promastigotes.ConclusionsWe present a validated easy-to-use affordable universal PCR assay to identify the most common Old World Leishmania species causing CL. This PCR assay could be used as a sensitive/specific technique to diagnose CL-causing Leishmania species in clinical samples with high accuracy.

Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex

ABSTRACTWe discovered that published polymerase chain reaction (PCR) assays for determining mating type (MAT) idiomorph failed to genotype some of the Fusarium fujikuroi species complex (FFSC) isolates recovered from Mangifera indica (mango), Swietenia macrophylla (big-leaf mahogany), Annona muricata (soursop), Bursera sp., and Tabebuia sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR–based diagnostic for typing MAT within the FFSC. To accomplish this objective, we mined the MAT1-1 or MAT1-2 locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the F. nisikadoi species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (SLA2) and apurinic endonuclease (APN1), the genes that flank the MAT locus in Fusarium. As expected, three genes were identified within MAT1-1 (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two in MAT1-2 (MAT1-2-1 and MAT1-2-9), using the ab initio prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting MAT1-1-2 and MAT1-2-1 successfully genotyped the entire 71-isolate validation panel, which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5–59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).

Isolation, characterization, and anthelminthic activities of a novel dichapetalin and other constituents of Dichapetalum filicaule

Context: Dichapetalum filicaule Breteler (Dichapetalaceae) is a rare species occurring only in Côte d’Ivoire and Ghana. Although research on several species of the genus has produced interesting bioactive compounds, particularly the Dichapetalins, a novel class of triterpenoids with antineoplastic properties, there is virtually no information on the ethnobotanical uses and chemical constituents of D. filicaule.Objective: The phytochemical and anthelminthic activities of the constituents of D. filicaule were investigated.Materials and methods: Chemical constituents of the petroleum ether, chloroform–acetone, and methanol root extracts of D. filicaule were isolated by column chromatography and characterized by their physico-chemical properties, 1-D and 2-D NMR spectroscopy and mass spectrometry. In vitro anthelminthic activity of the extracts and compounds against the human hookworm, Necator americanus, Stiles 1902 (Nematoda: Ancylostomatidae) was determined within a concentration range of 2500–250 μg/ml using the Egg Hatch Inhibition (EHI) Assay. The hookworm species were identified using a published polymerase chain reaction (PCR) method.Results: A new dichapetalin, dichapetalin X (1), together with the known dichapetalin A (2), pomolic acid (3), glycerol monostearate (4), D:A-friedooleanan-3β-ol (5), and D:A-friedooleanan-3-one (6) were isolated. Compounds 1, 2, and 4 exhibited EHI with IC50 values of 523.2, 162.4, and 306.0 μg/ml, respectively, against the hookworm. The positive control albendazole gave an IC50 value of 93.27 μg/ml.Discussion and conclusion: This is the first report of the phytochemical investigation of D. filicaule. The study has yielded a new dichapetalin and also demonstrated the potential anthelminthic properties of the constituents.

English

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is the most frequently used marker for distinguishing plant species. Previously published polymerase chain reaction (PCR) primers available for amplifying the ITS region from environmental plant samples provide a high degrees of success while maintaining a broad range of compatibility difficult to discriminate against fungal DNA. Thus, the plant-specific ITS primer design is required to achieve the preferential amplification of plant DNA with discrimination against fungal DNA from environmental plant samples. In this study, two primers, ITS1-F2 and ITS4-R, were newly designed and the specificity of these primers were tested against 14 accessions of Pulsatilla tongkangensis and 2 accessions of Pulsatilla cernua. Our results showed that ITS4-R, when paired with either a universal primer ITS5 or the newly designed primer ITS1-F2, efficiently amplified DNA from environmental P. cernua samples and discriminated against parasitic fungal DNAs, while another newly designed primer ITS1-F2, when paired with either a universal primer ITS4 or ITS4-R, could not preferentially amplify plant DNA or discriminate against fungal DNA. For P. tongkangensis, the dismatch of ITS5 primer resulted in noneffective or wrong amplification when paired with either ITS4-R or ITS4, while ITS1-F2, when paired with ITS4-R and ITS4, efficiently amplified plant DNA and both plant and fungal DNAs, respectively. This result suggested that the combination with ITS1-F2 and ITS4-R were intended to be specific to Pulsatilla species. This study will be particularly useful for detection and analysis of plant sequences from environmental or fungus-associated materials. Key words: Pulsatilla, nrDNA ITS, plant-specific primer design, fungus-associated plant discrimination, molecular identification.

Distinguishing human and possum faeces using PCR markers

Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers.

Absence of <emph type="ital">Helicobacter pylori</emph> in Pediatric Adenoid Hyperplasia

To (1) develop a reverse transcription-polymerase chain reaction assay to determine whether Helicobacter pylori and/or other members of the Helicobacteraceae family are detected in hyperplastic adenoids of children and (2) critically analyze published polymerase chain reaction methods to ascertain whether false-positive detection of H pylori has been reported. Cohort study. Adenoid biopsy specimens (78 hyperplastic and 15 normal) were collected from children aged 2 to 10 years. Total RNA was extracted before reverse transcription of bacterial RNA using Helicobacteraceae-specific primer. A nested reverse transcription-polymerase chain reaction protocol was designed to detect all species of the Helicobacteraceae family. A piece of each biopsy specimen was examined histologically. Laryngopharyngeal reflux was suspected in 41% of the children (n=23) on the basis of the Reflux Symptom Index. No evidence of H pylori was found in any adenoid sample. Candidatus Wolinella africanus was the only Helicobacteraceae family member detected in 1 hyperplastic adenoid. Histologic examination identified very few bacterial organisms. Previous polymerase chain reaction findings may be the result of false-positive H pylori detection. Inflammation and enlargement of the adenoids is not likely due to ongoing bacterial infection arising from laryngopharyngeal reflux. We conclude that H pylori and other Helicobacteraceae family members are not major contributors to the development of hyperplastic adenoids in children.

Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione transferases

Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.

Detection ofPseudomonas syringaepv.actinidiaeusing polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identifyPseudomonas syringaepv.actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains ofP. syringaepv.actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen,P. syringaepv.theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections asP. syringaepv.actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB,acnB,rpoD,pgiandcts) showed that none of these six strains was phylogenetically similar toP. syringaepv.actinidiae.In contrast to theP. syringaepv.actinidiaetype strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified asP. syringaepv.actinidiae. It was not possible to distinguishP. syringaepv.actinidiaefrom the phylogenetically similarP. syringaepv.theaeusing the ITS,gyrB,acnB,rpoD,pgiorctsgene regions to design PCR primers. BecauseP. syringaepv.theaeis unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus "Scalindua" group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.

Application of polymerase chain reaction–restriction fragment length polymorphism analysis and lab‐on‐a‐chip capillary electrophoresis for the specific identification of game and domestic meats

AbstractBACKGROUND: The objective of this study was to adapt and improve previously published polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods aimed at the identification of game and domestic meats, by replacing the gel electrophoretic steps for DNA fragment analysis by a chip‐based capillary electrophoresis system.RESULTS: PCR amplification of a mitochondrial 12S rRNA gene fragment and subsequent digestion of the amplicons with either MseI or a combination of MboII, BslI, and ApoI endonucleases generated characteristic PCR‐RFLP profiles that allowed discrimination among ten relevant game and domestic meat species. The Agilent 2100 Bioanalyzer utilises capillary electrophoresis on a microchip device that is capable of rapidly sizing DNA fragments, offering a valuable recent development for the analysis of complex DNA banding patterns.CONCLUSION: Results obtained in this work indicated that banding resolution on the system was sensitive, with detection of some small DNA fragments that were not observed with the published conventional PCR‐RFLP gel‐based method. Therefore, the new faster and easy handling procedure provides an additional powerful tool that can be employed for meat speciation. Copyright © 2009 Society of Chemical Industry

Use of Real-Time Quantitative PCR Targeting themsp2Protein Gene to Identify CrypticAnaplasma phagocytophilumInfections in Wildlife and Domestic Animals

Anaplasma phagocytophilum is an emerging pathogen throughout much of the Holarctic, where Ixodes spp. tick vectors occur. This organism was expected to be present at study sites in Humboldt County, north-western California, based on the presence of appropriate tick vectors, seropositivity in sentinel hosts, and previously reported human infections. However, despite high seroprevalence suggesting circulating A. phagocytophilum, active infections in dogs and wildlife (including suspected reservoir species) were rare using published polymerase chain reaction (PCR) protocols. This finding was possible if the published PCR protocol lacked sensitivity for strains in the study areas. We report a new TaqMan-PCR (TM-PCR) assay targeting the msp2 gene that has greater sensitivity and specificity for diverse A. phagocytophilum strains from this region. The new assay detected as few as one plasmid copy and a range of genetically diverse strains of A. phagocytophilum. Specificity was confirmed by failure to amplify targets of closely related bacteria. Application of the TM-PCR to samples from northern California confirmed PCR-positivity in 94 woodrats (71%; n=134), three (4%; n=80) bears, and seven (7%; n=97) domestic dogs. The msp2 TM-PCR protocol appears to be more sensitive for use in assays of samples from parts of western North America and possibly in other regions where populations are genetically diverse or divergent from eastern United States strains of A. phagocytophilum.

Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines

To determine relative detection rates and detection limits for 6 published polymerase chain reaction (PCR) assays used for detection of feline herpesvirus type 1 (FHV-1) DNA. 5 vaccines licensed for use in preventing FHV-1-associated disease; 15 conjunctival biopsy specimens collected from cats with keratitis, conjunctivitis, or both; and a plaque-purified field isolate of FHV-1 cultured in vitro. Vaccines and clinical samples were assessed for FHV-1 DNA by use of all 6 assays. Detection rates were calculated by assuming that any sample in which FHV-1 DNA was detected was a true-positive result. Detection limits were estimated by use of serial dilutions of DNA extracted from cultured FHV-1 and 1 clinical sample. Testing by use of all 6 assays resulted in detection of FHV-1 DNA in all 5 vaccines. Testing by use of all 6 assays yielded concordant results for 9 of 15 conjunctival biopsy specimens (8 with negative results and 1 with a positive result). Calculated detection rates for clinical samples ranged from 29% to 86%. Assay sensitivity was ranked similarly by use of detection rate or detection limit. Testing by use of all assays was equally likely to detect vaccine virus. Therefore, a positive PCR result in a cat may reflect vaccine virus rather than wild-type virus. Test sensitivity as assessed by detection limits and detection rates varied greatly. Because FHV-1 can be shed in clinically normal animals, high detection rate will not necessarily correlate with high diagnostic sensitivity.

PCR-Based Approach for Sequencing Mitochondrial Genomes of Decapod Crustaceans, with a Practical Example from Kuruma Prawn (Marsupenaeus japonicus)

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays.

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene ( hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR–restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.

Enumeration of Campylobacter in New Zealand recreational and drinking waters.

To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. These results suggest that water may act as a significant transmission route for human campylobacteriosis.

STRBase: a short tandem repeat DNA database for the human identity testing community.

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.

PCR-based CYP2D6 genotyping for Finnish lung cancer patients.

Polymorphism of the gene encoding for debrisoquine hydroxylase, i.e. CYP2D6, was determined genotypically for 122 healthy controls and 106 lung cancer patients using Xba I restriction fragment length polymorphism (RFLP) analysis, together with a combination of two recently published polymerase chain reaction (PCR) based approaches. Three different mutated alleles of the CYP2D6 gene were detected; CYP2D6B comprised 11.1% and 10.4% of the total alleles in the controls and in the lung cancer patients, CYP2D6A had frequencies of 5.7% and 2.8%, and CYP2D6D had frequencies of 3.3% and 2.4%, respectively. Only 17 of the 24 44 kb Xba I alleles (71%) were confirmed as defective alleles carrying the mutation in CYP2D6B loci, whereas all four 15 + 9 kb Xba I alleles contained the CYP2D6B mutation. Out of the 122 healthy controls, seven subjects (5.7%) were detected as poor metabolizers (PMs) of debrisoquine by the presence of two defective alleles, whereas only one PM genotype was found in the lung cancer patient group (0.9%). The reliability of this analysis was confirmed in a subgroup of the control subjects phenotyped by debrisoquine, where a perfect correlation between CYP2D6 phenotype and genotype was obtained. We observed no significant difference in the allelic frequencies between lung cancer patients with a history of heavy smoking and those who smoked less. However, statistical analysis showed a significant difference (p = 0.05) in distribution of the PM-associated genotypes between lung cancer patients (1/106) and healthy controls (7/122). This data thus supports the hypothesis that there is an increased risk of lung cancer for individuals who are extensive metabolizers of debrisoquine.