Direct PCR Assay Research Articles

Fast and sensitive detection of Trichophyton rubrum in superficial tinea and onychomycosis by use of a direct polymerase chain reaction assay

Detection of Trichophyton rubrum in superficial skin infections by conventional methods is time consuming and not always successful. However, with modern molecular methods, an alternative is in sight. The aim of this study was to compare the detection of T. rubrum by conventional methods and by a direct specific PCR assay under routine conditions. Skin scrapings (n = 464) and nail samples (n = 230) collected from suspected tinea lesions were equally divided for KOH-mounts, cultures and PCR-analysis. For the latter, DNA was extracted and PCR was performed with T. rubrum-specific primers. Of the scale samples, 16% were positive for T. rubrum in the culture and PCR as well, 9% were positive in the PCR only and 3% in the culture only, whereas 5% were only KOH-positive. The corresponding results for nail samples were 17%, 20%, 3% and 7%. PCR results were available after 2-5 days, culture results after 2-3 weeks. Our results show that a specific PCR assay can successfully be used to detect T. rubrum directly in samples collected from superficial skin lesions and nails under routine conditions. Compared with conventional methods, it is faster and more sensitive. We recommend its complementary use.

Application of direct PCR in a forensic case of yew poisoning

Intoxications with yew (Taxus spp.) pose a challenge to forensic toxicology because a variety of Taxus ingredients have been associated with its toxic effects. To provide preliminary evidence in cases where plant material is available, we introduce a novel direct PCR assay for the detection of DNA traces from Taxus spp. This assay has been successfully applied to a forensic case of suicidal poisoning via ingestion of Taxus leaves. PCR primers were designed to target a sequence located in the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA, which is well conserved among species of the genus Taxus and can, therefore, be exploited to discriminate between Taxus and other conifers. Because ITS1 exists as a multicopy sequence within the plant genome, the assay provides enough sensitivity to work with trace amounts that are below the DNA content of a single cell. Specificity of the assay was tested with DNA extracts from Taxaceae and selected representatives from other related plant families (Cephalotaxaceae, Cupressaceae and Pinaceae). When combined with the commercial Phire® Plant Direct PCR Kit (Finnzymes), the primers allowed application of a two-step cycling protocol (without the annealing step), and because direct PCR requires only little sample pre-treatment, results from PCR could be obtained within 1.5 h after analysis had begun. Direct PCR was performed with diluted gastric content from the forensic case. Amplification products of the expected size were purified and sequenced. Sequence data were subjected to Basic Local Alignment Search Tool analysis and affiliated with ITS1 from Taxus spp.

First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia.

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable.

Phytoplasma diseases of stone fruit trees in Turkey and their containment

Although fruit tree phytoplasmas were studied since 1999 in Turkey, there have been very limited studies and records on stone fruit tree phytoplasmas. The main symptoms reported on apricot, plum, peach and almond were chlorosis between veins, off season flowering and fruiting as a result of early bud breaking, longitudinal leaf rolling and quick die-back. More than 500 cultivated and wild Prunus plants in or nearby germplasm nurseries and commercial orchards during 2002–2009 were tested by using universal primers P1/P7 and fU5/rU3 in direct and nested PCR assays, respectively. Amplification products were digested with RsaI and SspI restriction enzymes. The average incidence of ‘Candidatus Phytoplasma prunorum’ was detected as 10.19%. The most infected stone fruit species were apricot and plum, followed by almond and peach. No phytoplasma was found in cherries and wild Prunus species.

Relapsed AML Patients with Mutational Shifts Harbor An Oligoclonal Primitive Cell Compartment at Presentation; Evidence for Clonal Selection towards Relapse

AbstractAbstract 751Previously we observed instability of type I/II mutations between diagnosis and relapse in a large portion (38%) of relapsed AML patients and this was associated with outcome (Bachas et al, Blood, 2010). Such instabilities may be explained by clonal selection during disease progression from oligoclonal initial AML populations. This hypothesis was tested in 6 AML patients with recurrent disease and mutational shifts between diagnosis and relapse. We determined the mutation profiles of mature and of immature subfractions of AML cells in initial diagnosis samples and related these to the corresponding relapse samples. Small amounts of cells (<50 cells) from mature and immature AML subfractions were sorted, followed by direct PCR assays for the detection of the frequently instable FMS like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD), Wilms Tumor 1 (WT1) and RAS mutations and the relatively stable Nucleophosmin 1 (NPM1) mutation. AML blasts were cell sorted based on CD45dim expression and primitive cell populations were defined by CD34/CD38 co-expression and refined based on the expression of CD133 and CD117. A lineage infidelity (Lininf) marker was used for designation of aberrant cells (Van Rhenen et al, Leukemia, 2007). Lymphocytes were sorted based on scatter properties and CD45bright expression and were regarded as wild type controls of primary patient material in mutation analyses. Immunophenotypes, cytogenetics and minimal residual disease (MRD) status were routinely assessed in our laboratory. Our results are indicative of an oligoclonal immature AML cell compartment at presentation: the immature CD45dimCD34+CD38dim/- compartments of initial samples were more heterogeneous according to their mutation profiles, when compared to the bulk of mature CD45dimCD34+ blast cells. Mutation profiles differed for at least one molecular marker among immature subfractions of the initial samples within 5 out of 6 patients. In the remaining patient, the mutation profiles were identical among sorted fractions of the initial sample. When comparing these data with the mutation profile of the bulk of AML cells of the relapse samples, in the latter patient and 2 out of the 5 above referred heterogeneous patients, mutational shifts could not be retraced to any subfraction of the initial sample. Strikingly, in the 3 other patients we found evidence for the clonal selection of a minor immature subclone of the initial sample, with a specific mutation profile that was predominantly present at relapse. In two of these patients we detected FLT3/ITD mutations of specific lengths, which were predominantly present at relapse, in a CD34+CD38−Lininf or CD34+CD38dimLininf subfraction of the initial sample. This ITD was undetectable in the bulk of AML cells by standard diagnostics. Similarly, in the other patient with clonal selection, the CD34+CD38−Lininf compartment of the initial sample was enriched for a WT1 exon 7 mutation that predominated at relapse, whereas no WT1 mutations were detected in the bulk of AML cells at presentation. In concordance with these observations, immunophenotype and MRD data also suggested a clonal selection in these patients. In the 3 patients that did show a mutational shift, but lacked direct indications for clonal selection, a de novo mutation may have been induced during therapy. Alternatively, the use of additional immunophenotypical and molecular markers may be required for these samples to identify the relevant subclones. The key finding of our study is that we could detect in the immature fraction of initial samples of 3 AML patients a rare and otherwise undetectable subclone with a distinctive mutational profile that was predominant in the bulk of AML cells at relapse. This has several clinical implications: risk group stratification based on the presence of mutations that are associated with prognosis can be refined by genetic characterization of oligoclonal immature AML subpopulations that may play a role in the development of relapse. In addition, early and accurate detection of these subclones will be crucial in the prevention of relapse by accurate timing and application of targeted treatment. In conclusion, our results underline the concept of oligoclonality in AML and the involvement of clonal selection of immature chemoresistant subclones in the development of relapse. This study was supported by the Dutch Cancer Society (VU2005-3666). Disclosures:No relevant conflicts of interest to declare.

Rapid and direct quantitative detection of viable bifidobacteria in probiotic yogurt by combination of ethidium monoazide and real-time PCR using a molecular beacon approach

The potential of ethidium monoazide (EMA) real-time PCR method based on molecular beacon probe for rapid detection of viable bifidobacteria present in probiotic yogurt was evaluated in this work. A real-time PCR with molecular beacon assay was developed to determine genus Bifidobacterium quantitatively in order to increase the sensitivity and specificity of assay. EMA was used to treat probiotic yogurt prior to DNA extraction and real-time PCR detection to allow detection of only viable bacteria. The primer set of Bif-F/Bif-R which is genus-specific for Bifid. was designed. The specificity of the probes ensures that no signal is generated by non-target amplicons. Linear regression analysis demonstrated a good correlation (R² = 0·9948) between the EMA real-time PCR results and the plate counting, and real-time quantitative PCR results correlated adequately with enumeration of bifidobacteria by culture for commercial probiotic yogurt. This culture-independent approach is promising for the direct and rapid detection of viable bifidobacteria in commercial probiotic yogurt, and the detection can be carried out within 4 h. The detection limit for this method is about 10⁴ cell/ml. In conclusion, the direct quantitative EMA real-time PCR assay based on molecular beacon described in this research is a rapid and quantitative method.

A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep

A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay ( Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.

First report of a phytoplasma associated with Periploca aphylla witches’ broom in Iran

Periploca aphylla (Asclepiadaceae), is a large erect branched shrub, grown on different altitudes, in desert or in dry, insolated grounds, on rocky and sandy slopes throughout Southwest Asia. Periploca aphylla is commonly used for feeding camels and goats. Its latex is applied as a poultice in tumours and swellings, while the decoction of the bark is used as a purgative or against fever (Kazimierz, 1966). In September 2008, symptoms of witches’ broom, proliferation and shoot dieback were observed on P. aphylla in Hormozgan province, Iran. The affected plants developed dense clusters of highly proliferating branches with shortened internodes. Total DNA was extracted separately from 0·5 g of shoots of ten symptom-bearing and five symptomless plants using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1990). The samples were analysed for phytoplasma DNA by both direct and nested PCR assays using phytoplasma universal 16S rDNA gene primer pairs P1/P7 and R16F2n/R16R2 that amplify fragments of 1800 bp and 1250 bp, respectively. A 1250 bp PCR product was amplified with nested PCR from all symptom-bearing, but not from symptomless, plants. Two amplicons were cloned using cloning commercial kit #K1214 (Fermentas), and sequenced. The partial 16S rDNA sequence (GenBank Accession No. GQ249159) of the phytoplasma associated with P. aphylla witches’ broom was most closely related (98% sequence identity) to salt cedar witches’ broom phytoplasma, ‘Candidatus Phytoplasma tamaricis’ (FJ432664) (Zhao et al., 2009). Moreover, P. aphylla witches’ broom phytoplasma showed a 96% sequence identity with strains of group 16SrX, apple proliferation, including a ‘Ca. Phytoplasma pyri’-related strain from subgroup 16SrX-C, previously associated with pear decline in Iran (Salehi et al., 2008), which has been reported in almost all pear growing areas of Fars, Isfahan, Tehran and Khorasan provinces. This is the first report of a phytoplasma associated with P. aphylla witches’ broom.

Molecular Screening, Isolation, and Characterization of Enterohemorrhagic Escherichia coli O157:H7 from Retail Shrimp

Foodborne outbreaks attributed to the contamination of foods with enterohemorrhagic Escherichia coli (EHEC) O157:H7 are a growing global concern. Fish and shrimp samples obtained from different retail fish markets in Cochin, India, were screened by direct PCR assays targeting three important virulence markers of EHEC, the intimin protein (eaeA gene), enterohemolysin (hlyA gene), and Shiga toxin (stx gene). One shrimp (Fenneropenaeus indicus) sample was positive for all these virulence markers, and seven typical E. coli O157:H7 isolates were recovered from the marker-positive shrimp sample. This is the first report of recovery of typical E. coli O157:H7 from fish or shellfish in India. All the typical EHEC isolates had a characteristic reaction in eosin methylene blue agar and belonged to IMViC (indole, methyl red, Voges Proskauer, Simmons citrate reactions) biotype I. These isolates also were negative for sorbitol and methylumbelliferyl-beta-glucuronide and exhibited beta-hemolytic activity. One isolate showed self-agglutination for E. coli O157 antisera and produced a false-positive reaction with CHROMagar O157. These typical EHEC isolates belonged to a restricted biotype group and had a very low multiple antibiotic resistance index. Isolation of E. coli O157:H7 in fish and shellfish indicates that strict adherence to hygienic handling methods and proper cooking or processing is needed before consumption of these products.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

BackgroundActivating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.Methodology/Principal FindingsWe determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.ConclusionsMASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs

To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV). 22 commercially available M hyopneumoniae bacterins. Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe. Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays. Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Monitoring of plasmid dissociation and pathogenic potential amongYersinia enterocolotica andYersinia pseudotuberculosis during storage of refrigerated pork meat

To determine the pathogenic potential ofYersinia in pork meat samples, different strains ofYersinia enterocolitica andYersinia pseudotuberculosis were inoculated on pieces of raw pork meat samples and exposed to +4 °C and −20 °C for 90 days. The effect of temperature, pH, total bacterial number, and inoculation dose of meat samples on virulence plasmid dissociation among different serotypes ofY. enterocolitica andY. pseudotuberculosis was monitored by using modified cultural media and confirmed by PCR assay andin vivo mouse model. In comparison with the intial dose of contamination,Yersinia cells from the less pathogenic serotypes (O:3 and O:9 ofY. enterocolitica) survived better than the high pathogenic serotypes ofY. enterocolitica O:8 andY. pseudotuberculosis O:3. Maximal rates for plasmid dissociation were found among the cells of the less pathogenic serotypes O:3 and O:9 ofY. enterocolitica. In parallel, at each time point highly virulent phenotypes ofYersinia were proved by direct PCR assay including detection of both chromosomal (ail) and plasmid (virF) genes. The real pathogenic potential ofYersinia cells occurred after plasmid dissociation during long time cold storage was confirmed in a newly designed mouse model mimicking the immunosupression status of humans. Therefore, the impact of refrigeration temperatures differs for theYersinia serotypes and modulates their pathogenic potential resulting in a risk factor for human health.

First Report of 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Safflower Phyllody Disease in Iran.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT-3'/5'-CGTCCTTCATCGGCTCTT-3'), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG-3'/5'-TGACGGGCGGTGTGTACAAACCCCG-3') yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members ('Candidatus Phytoplasma trifolii') and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the 'Ca. Phytoplasma trifolii' reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a 'Ca. Phytoplasma trifolii'-related strain.

XMAN: extreme MApping of OligoNucleotides

BackgroundThe ability to rapidly map millions of oligonucleotide fragments to a reference genome is crucial to many high throughput genomic technologies.ResultsWe propose an intuitive and efficient algorithm, titled extreme MApping of OligoNucleotide (xMAN), to rapidly map millions of oligonucleotide fragments to a genome of any length. By converting oligonucleotides to integers hashed in RAM, xMAN can scan through genomes using bit shifting operation and achieve at least one order of magnitude speed increase over existing tools. xMAN can map the 42 million 25-mer probes on the Affymetrix whole human genome tiling arrays to the entire genome in less than 6 CPU hours.ConclusionsIn addition to the speed advantage, we found the probe mapping of xMAN to substantially improve the final analysis results in both a spike-in experiment on ENCODE tiling arrays and an estrogen receptor ChIP-chip experiment on whole human genome tiling arrays. Those improvements were confirmed by direct ChIP and real-time PCR assay. xMAN can be further extended for application to other high-throughput genomic technologies for oligonucleotide mapping.

Molecular Detection of Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' Infection in Cats by Direct PCR Using Whole Blood without DNA Extraction

Detection of hemotropic Mycoplasma spp. infection was attempted in cats by PCR using whole blood without DNA extraction. A total 46 of 54 (85%) cats with suspected Mycoplasma spp. infection showed a positive reaction, corresponding completely with the results of standard PCR testing. The direct PCR assay was sensitive enough to detect more than 0.0061% parasitemia for ;C. M. haemominutum' and 0.0075% parasitemia for M. haemofelis. These data indicate that the direct PCR assay might be sufficient for use as a tool in clinical examinations.

Semi-nested polymerase chain reaction for detection of toxigenic Vibrio cholerae from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10(3) CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10(4) CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.

Heparanase and Survivin—potential markers of aggressiveness of colon cancers

14524 Background: Survivin is an inhibitor of apoptosis and specifically expressed in several human cancers. Heparanase seems to play an important role in cancer metastasis, angiogenesis and regulation of COX-2 expression. The purpose of this pilot study was to evaluate whether there is a difference in the level of survivin and heparanase expression in paraffin-embedded tissue from patients with colon cancer with and without metastasis. Methods: Paraffin-embedded tissue samples were obtained from 32 patients with primary colon tumors with (n = 15) and without liver metastases (n = 17) at the time of diagnosis. After laser capture microdissection direct quantitative real-time reverse transcriptase PCR (RT-PCR, TaqMan™) assays were performed in triplicates to determine survivin and heparanase gene expression levels. Gene expression was normalized with beta-Actin. Decision tree analysis was used to assess whether higher levels of these genes are associated with the existence of metastasis. Results: Neither survivin nor heparanase gene expression alone showed a significant relationship with the pM-Stage, although the detection levels of heparanase showed a tendency towards significant correlation with the existence of liver metastasis (p = 0.06). Decision tree analysis was used to split patients into two groups based on cut-off values of gene expression most closely associated with the pM-stage. The groupings determined by this analysis were significantly differently correlated with metastasis by Spearman’s test (p=0.03). Conclusions: Our results show that high expression levels of both survivin and heparanase in paraffin- embedded tissue are significantly correlated with the pM-Stage in this patient cohort. In conclusion, using intratumoral survivin and heparanase gene expression levels might be useful to identify patients at risk for liver metastasis. Prospective and larger clinical studies are warranted to validate our preliminary findings. No significant financial relationships to disclose.

Prevalence of Mycoplasma bovis in backgrounding and stocker cattle operations

To characterize the prevalence of Mycoplasma bovis infection in backgrounding and stocker cattle operations and compare bacteriologic culture with PCR assay for detection of M bovis. Prospective descriptive study. 432 calves, 3 to 9 months old, from 9 operations. 2 nasal swab specimens were collected from each calf. Swab specimens were evaluated via bacteriologic culture and PCR assay for organisms of the class Mollicutes and M bovis. Culture results were considered negative if no growth occurred within 21 days. Positive results were indicated by characteristic colony formation with PCR assay confirmation. Deoxyribonucleic acid was extracted from 1 swab specimen for direct PCR assay for Mollicutes and M bovis. Of 432 calves, 374 (87%) had positive results for Mollicutes via PCR assay and 63 (15%) via culture. Seven (2%) calves had positive results for M bovis via PCR assay and 10 (2%) via culture. Prevalence of Mollicutes at the farm level ranged from 54% to 100% via PCR assay and from 0% to 59% via culture. Prevalence of M bovis at the farm level ranged from 0% to 4% via PCR assay and from 0% to 6% via culture. Calves that shed M bovis were significantly more likely to have a fever than were calves that did not shed M bovis. M bovis was detected at a low level in recently purchased backgrounded and stocker calves in Georgia. Although slightly more infected calves were detected via culture and PCR assay together, PCR assay appeared to accurately identify M bovis at the farm level.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction

A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.