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.     

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using promoter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present within the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Importance of the serovar-specific plasmid for virulence of salmonella strains in calves

To evaluate the influence of serovar-specific plasmids on salmonella virulence in calves, experiments were performed involving infection, by the oral route, with mixtures of strains containing equal counts of a plasmid-carrying and a plasmid-free strain of the same serovar. The concentration ratio between the plasmid-carrying and the plasmid-free strain which had developed in the organs of the infected animals was used for a comparative evaluation of virulence and pathogenetic behaviour of the strains. While in the S. typhimurium strains studied, the presence of the plasmid was accompanied by a significantly increased colonization and multiplication of the agent in the host's body, examination of S. enteritidis and S. dublin revealed that the plasmid-free strains exhibited identical or even significantly higher bacterial counts than the plasmid-carrying strains in organs. The fact that plasmid-free salmonella strains with a high virulence for calves have been found demonstrates that the presence of a serovar-specific plasmid is not an indispensable requirement for the development of salmonellosis in calves.     

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.     

The possibility of using highly disperse iron for the directed transport of thyroxin in the body

In order to isolate promoters of mouse TGF-beta receptor genes, we used inverse PCR with highly overlapped primers corresponding to the 5' sequence of the receptor cDNAs. Nested primer sets only covered a 30- to 40-base region of the sequences. HinfI-digested and self-ligated mouse genomic DNA was used as a PCR template. Only one band for each receptor was seen after PCR. The amplified DNA fragments could direct luciferase production when the luciferase coding sequence was ligated after the fragments. The sequence of the fragment which correspond to the type II receptor showed partial homology with the promoter region of the human TGF-beta type II receptor. Thus, the inverse PCR with highly overlapped primers could be an easy way to isolate the promoter regions of many genes.     

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.     

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.     

Partial inhibition of amplifications by primers of EHEC genes

Diagnostic value of multiplex polymerase chain reaction (PCR) was examined by using three primer pairs, specific for the common conserved region of stx1 and stx2, eae and an enterohaemolysin A gene (ehxA). The sensitivity in respect of each amplicon decreased with three exponents comparing to the individual PCR reactions. These PCR reactions were partially inhibited by the presence of certain additional primers. This inhibitory effect was template-concentration dependent, and was partially balanced by usage of increased amount of dNTP. Taq DNA polymerase in a range of 0.3-1.25 U/reaction did not influence the inhibition. The same inhibition was detected if the annealing temperature was changed from 48 degrees C to 57 degrees C. Pairs of EHEC primers inhibited a Salmonella enteritidis virulence-plasmid specific gene amplification, as well. Theoretical inhibiting effects were predicted by Primer Premier software but our observations can be sufficiently explained neither by the competitions between the specific and aspecific amplifications nor by the inhibition caused by dimerization of primers.     

Cloning and assembly of PCR products using modified primers and DNA repair enzymes

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

Time-course effects of human recombinant luteinizing hormone on porcine Leydig cell specific differentiated functions

Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.     

Group specific PCR-detection of potential trichothecene-producing Fusarium-species in pure cultures and cereal samples

A PCR based assay (Tox5 PCR) which analyses Fusarium species potentially producing trichothecenes was developed using a pair of primers derived from the DNA-sequence of the trichodiene synthase gene (tri5). The primer pair was tested using DNA isolated from a variety of strains representing 64 species and varieties of Fusarium as well as from other fungi, bacteria and cereals. A 658 bp PCR fragment was specifically amplified with DNA isolated from strains of species belonging to the Fusarium sections Discolor, Sporotrichiella, Arthrosporiella, Gibbosum, and "Dlaminia". PCR products obtained were sequenced. Alignment to tri5 sequences given in the literature revealed a high degree of homology. Results of the PCR developed correlated well with literature data on the trichothecene producing capabilities of the respective species. Potential trichothecene producing fusaria were detected in contaminated cereals and malts using the Tox5 PCR assay. Intensity of the signals produced were well correlated with the concentration of deoxynivalenol (DON) in samples of wheat.     

In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR

The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125-and 147-bp DNA fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.     

Cost minimization analysis of various treatment options for surgical stage I endometrial carcinoma

A rapid bioluminometric technique for real-time detection of known single-base changes is presented. The concept relies on the measurement of the difference in primer extension efficiency by a DNA polymerase of a matched over a mismatched 3' terminal. The rate of the DNA polymerase-catalyzed primer extension is measured by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (P. Nyrén (1987) Anal. Biochem. 167, 235-238). The PPi formed in the polymerization reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the single-base detection assay, immobilized single-stranded DNA fragments are used as template. Two detection primers differing with one base at the 3' end are designed, one precisely complementary to the nonmutated DNA sequence and the other precisely complementary to the mutated DNA sequence. The primers are hybridized with the 3'-termini over the base of interest and the primer extension rates are, after incubation with DNA polymerase and deoxynucleotides, measured with the ELIDA. We show that the relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini. The possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Phage restriction and the presence of small plasmids in Salmonella enteritidis

Sequence variation within the variable region of the 16S rRNA at position 440 to 480 allowed the synthesis of specific PCR primers for the identification of groups within the species Photorhabdus luminescens, symbionts of entomopathogenic nematodes of the genus Heterorhabditis. For the second PCR primer the highly conserved region at 755 to 795 was used. The P. luminescens type strain specific primer could not recognize any other P. luminescens strain. The primer TEMPERATUS based on the sequence of strain DSM12190 (isolated from North West European H. megidis strain HSH2) identified all P. luminescens associated with H. megidis from North West Europe and two isolates from closely the related nematode strains from Ireland. The primer TROPICUS based on strain DSM12191 (isolated from the nematode type strain H. indica strain LN2) identified P. luminescens of tropical origin isolated from H. indica. Symbionts of H. bacteriophora could not yet be separated into well described groups with the primers used. A comparison of sequence data resulted in the identification of additional groups. The non-symbiotic P. luminescens isolates are distinct in the variable region. The group HELIOTHIDIS contains 15 P. luminescens associated with H. bacteriophora from North East America. The MARELATUS group contains symbionts of the nematode H. marelatus from the West Coast of the US. The data together with the specific symbiotic association of P. luminescens strains with different nematode species support the division of the taxon P. luminescens into different species.     

Identification of new verocytotoxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates

The sequence of a verocytotoxin 2 (VT2) variant gene that was untypeable by the B subunit PCR and restriction fragment length polymorphism analysis (PCR-RFLP) method described by Tyler et al. (S. D. Tyler, W. M. Johnson, H. Lior, G. Wang, and K. R. Rozee, J. Clin. Microbiol. 29:1339-1343, 1991) was determined and compared with published sequences. It was highly homologous to two recently reported VT2 variant sequences. The PCR-RFLP method described by Tyler et al. was extended to include these new sequences. New VT2 variants were identified in 65 of 359 VT-producing Escherichia coli (VTEC) with newly designed primers (VT2-cm and VT2-f) and were characterized as well by restriction analysis of the amplification products obtained with another VT2-specific primer pair (VT2-e and VT2-f). The VT genes harbored by 64 of these isolates proved to be untypeable by Tyler's PCR-RFLP method because no amplification was obtained with the primers used with this method (VT2-c and VT2-d). The last isolate harbored the new variant gene in addition to VT2vh-a. None of the isolates harboring these new toxin genes belonged to serogroups O157, O26, O103, O111, and O145. All 65 isolates were negative for the eaeA gene and were significantly less frequently enterohemolytic or positive for the enterohemorrhagic E. coli (EHEC) virulence plasmid than non-O157 VTEC isolates harboring other VT2 genes. They were also less frequently isolated from patients with EHEC-associated symptoms. The extended PCR-RFLP typing method is a useful tool to identify less-virulent VTEC isolates and for VT genotyping in epidemiological studies with non-O157 strains.     

Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA

A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.