High-dose chemotherapy supported by peripheral blood stem cells to treat intermediate--and high-grade non-Hodgkin''s lymphomas

The presence or absence of the mecA gene, the determinant of resistance to all beta-lactam antibiotics, was examined in clinical isolates of Staphylococcus aureus by multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bp nuc- based PCR fragment (amplification product of the nuc gene encoding specific Staphylococcus aureus nuclease) and a 533-bp mecA-based PCR fragment (amplification product of the mecA gene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.     

A universal approach to bacterial molecular epidemiology by polymerase chain reaction ribotyping

Oligonucleotide primers complementary to conserved regions of the 16S and 23S ribosomal RNA genes were used to amplify the 16S-23S intergenic spacer region of bacterial pathogens. The amplification patterns produced were compared for their potential use in molecular epidemiologic analysis. This method, polymerase chain reaction (PCR) ribotyping, was applied to isolates of Staphylococcus aureus, Enterococcus faecium, Escherichia coli, and Enterobacter species. Length polymorphisms in the amplified DNA distinguished unrelated strains of all bacteria. The banding patterns of 3 S. aureus isolates from the blood of 1 patient on 3 consecutive days were identical. Plasmid analysis, biotyping, and antibiograms were also obtained on the Enterobacter isolates. All three of these methods showed considerable variability after in vitro passage of bacteria, but PCR ribotypes remained stable. Results demonstrate the utility of the conserved primers for PCR ribotyping, a widely applicable method for the molecular epidemiology of genetically diverse bacteria.     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

Detection and identification of Actinobacillus pleuropneumoniae serotype 5 by multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10(2) CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Cloning and nucleotide sequence of a specific DNA fragment from Paracoccidioides brasiliensis

We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.     

Fot 1 insertions in the Fusarium oxysporum f. sp. albedinis genome provide diagnostic PCR targets for detection of the date palm pathogen

Populations of Fusarium oxysporum f. sp. albedinis, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar Fot 1 hybridization patterns. In order to develop a sensitive diagnostic tool for F. oxysporum f. sp. albedinis detection, we isolated several DNA clones containing a copy of the transposable element Fot 1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several Fot 1 insertion sites. When tested on a large sample of Fusarium isolates, including 286 F. oxysporum f. sp. albedinis isolates, 17 other special forms, nonpathogenic F. oxysporum isolates from palm grove soils, and 8 other Fusarium species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in F. oxysporum f. sp. albedinis. Sequence analysis showed that one of the Fot 1 copies was truncated, lacking 182 bp at its 3' terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the Fot 1 truncated copy and its 3' site of insertion in the F. oxysporum f. sp. albedinis genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for F. oxysporum f. sp. albedinis isolates.     

Investigation of film curing stages by dielectric analysis and physical characterization

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.     

Genetic heterogeneity in human T-cell leukemia/lymphoma virus type II

DNA from the peripheral blood mononuclear cells of 17 different individuals infected with human T-cell lymphoma/leukemia virus type II (HTLV-II) was successfully amplified by the polymerase chain reaction (PCR) with the primer pair SK110/SK111. This primer pair is conserved among the pol genes of all primate T-cell lymphoma viruses (PTLV) and flanks a 140-bp fragment of DNA which, when used in comparative analyses, reflects the relative degree of diversity among PTLV genomes. Cloning, sequencing, and phylogenetic comparisons of these amplified 140-bp pol fragments indicated that there are at least two distinct genetic substrains of HTLV-II in the Western Hemisphere. These data were confirmed for selected isolates by performing PCR, cloning, and sequencing with to 10 additional primer pair-probe sets specific for different regions throughout the PTLV genome. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-II, while the prototype MoT isolate defines the original substrain. There was greater diversity among HTLV-II New World strains than among HTLV-I New World strains. In fact, the heterogeneity among HTLV-II strains from the Western Hemisphere was similar to that observed in HTLV-I and simian T-cell lymphoma/leukemia virus type I isolates from around the world, including Japan, Africa, and Papua New Guinea. Given these geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV, these data suggest either that HTLV-II has existed for a long time in the indigenous Amerindian population or that HTLV-II isolates introduced into the New World were more heterogeneous than the HTLV-I strains introduced into the New World.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.     

Typing Neisseria meningitidis by analysis of restriction fragment length polymorphisms in the gene encoding the class 1 outer membrane protein: application to assessment of epidemics throughout the last 4 decades in China

A typing method was developed for Neisseria meningitidis serogroup A by analysis of restriction fragment length polymorphisms (RFLP) of the class 1 outer membrane protein gene (porA). By using appropriate primers, an approximately 1,116-bp fragment of the porA gene was amplified by PCR and then was digested with the restriction endonuclease MspI. The digestion products were separated on 10% polyacrylamide gels and were stained with silver. One hundred three clinical isolates of group A N. meningitidis from 17 provinces of China collected over a 26-year period were analyzed. Results of MspI-generated RFLP profiles of PCR-amplified porA genes were compared with those obtained by conventional serosubtyping. There was a band of about 400 bp common to all strains examined, and the 103 strains of serogroup A resulted in 22 unique RFLP patterns. The differences in bands could be observed mainly in the range of 120 to 280 bp. The smaller fragments were useful in distinguishing meningococci with the same serosubtype. Three epidemic periods were characterized by the presence of three distinct genotypes (a1, a2, and a3), accounting for 74.5% of the strains examined (3.88, 26.21, and 44.66%, respectively). Three predominant RFLP patterns were correlated epidemiologically with cycles of epidemic meningococcal meningitis and were well-matched to the predominant serosubtypes (P1.9, P1.7, 10, and P1.9) that presented at the same prevalence cycles. The genotyping yielded information that allowed strains from one epidemic to be distinguished from those from another that would have been indistinguishable if only serotyping and serosubtyping were available. Therefore, the PCR-RFLP typing method was very useful in the epidemiologic investigation of group A meningococcal meningitis.     

Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.     

Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.     

In vitro studies of pharmacodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2x to 64x the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log10 CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10x the MIC of vancomycin with two different half-lives (t1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log10 CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t1/2 of 5 h (approximate terminal t1/2 in humans) was used. The PMEs at t1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.     

Intergenic transcribed spacer PCR ribotyping for differentiation of Saccharomyces species and interspecific hybrids

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.     

A contingency-based approach for assessing policies on aging

The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.     

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.     

Nucleic acid amplification for rapid detection of Rhodococcus equi in equine blood and tracheal wash fluids

OBJECTIVE: To evaluate the ability of nucleic acid amplification techniques to detect Rhodococcus equi in equine buffy coat, blood, and tracheal wash fluid and to differentiate between virulent and avirulent strains of the bacteria. SAMPLE POPULATION: Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses. PROCEDURE: Logarithmic dilutions of virulent and avirulent strains of R equi were added to equine buffy coat and tracheal wash fluid samples. The DNA was extracted and amplified by polymerase chain reaction (PCR), using primers specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi. RESULTS: PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasmid primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacteria. Virulent strains of R equi could be identified by PCR and differentiated from avirulent strains within 12 to 24 hours after sample collection, with as few as 10 to 100 organisms present. CONCLUSIONS: PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and can differentiate between virulent and avirulent strains of the organism. CLINICAL RELEVANCE: Because PCR can confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination.