A PCR-microplate capture hybridization method to detectListeria monocytogenesin blood

In order to improve the diagnosis ofListeria monocytogenesinfection, we have developed a polymerase chain reaction (PCR)-based assay combined with microplate capture hybridization technique. The system is based on selective amplification ofL. monocytogeneswith two specific primers based on theiapgene. The amplicon produced, with digoxigenin 11-dUTP incorporated during PCR, is hybridized in streptavidin-coated microtitre plates prepared with biotinylated specific DNA probe. The method involved requires approximately 6–8 h, and its high sensitivity, rapidity and simplicity should make it valuable for diagnosis and for epidemiological studies of listeriosis.     

Simultaneous detection ofListeriaspp. andListeria monocytogenesby reverse hybridization with 16S–23S rRNA spacer probes

Enzymatic amplification results showed thatListeriaspecies have at least two 16S–23S rRNA spacer regions of different lengths. These spacer regions ofL. monocytogenes,L. ivanoviiandL. seeligeriwere cloned after enzymatic amplification. Sequence analysis of the inserts revealed two spacers of 245–246 bp and 496–498 bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. OneListeriaspp.-specific probe, LIS-ICG4, was deduced from the 245-bp spacer and aL. monocytogenes-specific probe, LMO-ICG5, was inferred from the 496-bp spacer. The specificity of both probes was tested in a reverse hybridization assay (Line Probe Assay, LiPA). Both LIS-ICG4 and LMO-ICG5 proved to be highly specific when hybridized to a large collection ofListeriastrains and strains from other relevant taxa. The LiPA test herein described for the simultaneous detection ofListeriaspp. andL. monocytogenescan be expanded to detect other foodborne pathogens.     

Detection ofPlasmodium ovalemalaria parasites by species-specific 18S rRNA gene amplification

A polymerase chain reaction (PCR) assay was developed for the specific detection ofPlasmodium ovale, one of the four malaria parasites that infect humans. On the basis of sequence variation of thePlasmodium18S ribosomal RNA (rRNA) gene, oligonucleotide primers for PCR were designed to amplify various fragments of theP. ovalegene. Using a recombinant plasmid with the completeP. ovale18S rRNA gene as target, 59 primer combinations were tested so that at least one of the pairs was species-specific while the other primer was either genus conserved orP. ovalespecies-specific. Three primer pairs yielding DNA fragments at stringent conditions were further tested against genomic DNA of four human malaria species. This approach yieldedP. ovalespecies-specific primer pairs that may be useful for further field testing.     

Development of internal controls for PCR detection ofBacillus anthracis

This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection ofBacillus anthracisstrains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes ofB. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences ofBacillus cereuswere then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.     

Development and clinical application of a polymerase chain reaction-based assay for detectingBacteroides ureolyticus

We developed a polymerase chain reaction (PCR)-based assay for detectingBacteroides ureolyticus. No DNA fragments hybridizing the internal probe specific forB. ureolyticus were amplified from the DNAs of other bacterial species tested. This assay can detect the amount of DNA corresponding to 18 cells ofB. ureolyticus. Using this assay, we examined urethral swab specimens from men with nongonococcal urethritis (NGU) and asymptomatic men for the presence ofB. ureolyticus. WhileB. ureolyticus was detected in 6 (12.0%) of 50 asymptomatic men, this was not significantly different from the detection rate of 19.3% (22 of 114 specimens) in men with NGU. This study demonstrates that the PCR-based assay can be applied for detectingB. ureolyticus in clinical specimens and suggests that this assay might develop into a useful tool for analyzing the pathogenicity of this organism in various organs.     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: use of a PCR-DNA enzyme immunoassay

A PCR-DNA enzyme immunoassay (PCR-DEIA) was developed for identification of the coagulase-positive species Staphylococcus intermedius. Two PCR primers and a hybridization probe were designed to target specific sequences of the S. intermedius thermonuclease (nuc) gene. In addition to S. intermedius reference strains, the PCR-DEIA was tested using 295 veterinary and human S. intermedius isolates. A specific 933-bp DNA fragment was successfully amplified in 281 (94.9%) S. intermedius isolates. Five canine isolates showed an unexpected 2.8-kbp band. Except for 10 amplicons derived from equine, camel, and pigeon isolates, all positive PCR results (n = 288, 96.6%) were confirmed by the colorimetric microtiter plate DEIA hybridization. Isolates that failed both in amplification and DEIA hybridization were only observed in equine isolates (10/23, 43.5%). Except for the limitations with isolates of hoofed animals, the S. intermediusnuc PCR assay has potential for rapid identification of S. intermedius and differentiation from other coagulase-positive staphylococci including S. aureus.     

Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs

We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in aSpiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10⁴to 10⁸M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process.     

Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5′ nuclease PCR assays

This study describes fluorogenic 5′ nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi andE.intestinalis . The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10⁴ and 10⁻¹ spores per PCR sample. The coefficients of variation were ≤5·2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10⁴ spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.     

Rapid and sensitive detection ofChlamydia trachomatisusing a ligatable binary RNA probe and Qβ replicase

A simple assay format was developed for the direct detection ofC. trachomatisrRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 10⁴targets) was suppressed by blocking sequences in the 5′ MDV reporter probe fragment complementary to the 3′ fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid–selection–amplification protocol, yielded a low level of assay background which was reduced to <2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 10³molecules of 23S rRNA (>95% responding) and could detect a single elementary body (EB) ofChlamydia trachomatisor 1–10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10⁵-fold range, permitting the determination of target level within an order of magnitude. The assay showed 10⁹-fold discrimination overChlamydia pneumonae(TWAR) rRNA. High levels of culturedC. albicans,E. coli,S. aureus, orN. gonorrhoeaehad no detectable effect on assay background or the ability to detect a single elementary body.     

Detection of parvovirus B19 by dot-blot and polymerase chain reaction

Our studies compare detection of parvovirus B19 DNA by dot-blot hybridization and by the polymerase chain reaction (PCR). Compared to detection by dot-blot hybridization with a 32P-oligonucleotide probe, the sensitivity of PCR product detection by ethidium bromide fluorescence is not compromised when the size of the amplification target and number of cycles of amplification are properly selected. Our PCR assay simultaneously amplifies two targets in the B19 genome and yields a sensitivity 10 times greater than 32P-labelled oligonucleotide--dot-blot hybridization (dot-blot). In 126 serum samples from cases of suspected parvovirus B19 infection, viral DNA was detected by PCR in 17% (21/126), whereas dot-blot detected B19 DNA in 0.8% (1/126). Parvovirus B19-specific antibodies were detected in 69% (87/126) of the suspected clinical cases by immunoblot. Nineteen of the antibody-positive specimens were DNA positive of which three were IgM-positive/IgG-negative, 11 IgM-positive/IgG-positive and five IgM-negative/IgG-positive. The only dot-blot DNA positive sample was also PCR-DNA positive but was B19 IgM-negative/IgG-negative. Eighty-six B19 antibody-negative, clinically uninfected controls were also negative for B19 DNA by PCR and by dot-blot. These studies confirm the increased sensitivity of gene amplification by PCR for detection of B19 DNA and demonstrate that two targets in B19 DNA can be amplified simultaneously with resultant increased ease of interpretation. Finally, detection of the two B19-specific products by ethidium bromide fluorescence is documented.     

Rapid assay for detection of methicillin-resistantStaphylococcus aureususing multiplex PCR

The presence or absence of themecAgene, the determinant of resistance to all β-lactam antibiotics, was examined in clinical isolates ofStaphylococcus aureusby multiplex polymerase chain reaction (MPCR). Two pairs of primers were used, which yielded two specific products; a 280-bpnuc-based PCR fragment (amplification product of thenucgene encoding specificStaphylococcus aureusnuclease) and a 533-bpmecA-based PCR fragment (amplification product of themecAgene). The MPCR system was designed to be incorporated into the work flow in clinical diagnostic laboratories as a routine analysis.     

A novel male-specific DNA sequence in the common carp, Cyprinus carpio

In this study, we identified a sex-specific DNA marker using randomly amplified polymorphic DNA (RAPD) fingerprinting in Yellow River carps (Cyprinus carpio from the Yellow River). Two hundred and twenty random primers were used in pooled DNA samples and individual DNA samples from male and female fish for RAPD fingerprinting. When using the primer S2107, a novel sex-specific PCR product was identified in all male individuals. DNA sequencing revealed that this 909 bp long DNA fragment has a low similarity to a repetitive sequence in zebrafish Danio rerio. In order to confirm the amplification results, two primers were designed within the male-specific sequence in order to amplify the sex-specific fragment from genomic DNA of male and female carps for sexing by PCR. The results indicated bands specific for males but not females. Respective results were obtained in dot blot and Southern blot hybridization experiments when using this sex-specific fragment as the probe. The sex-specific pattern was observed in 30 individuals from three separate common carp stocks, suggesting that the sequence is conserved in common carp species. However, no hybridization signals were found in grass carps Ctenopharyngodon idella, which are related to common carps. We submit to use this sex-specific fragment as a marker to rapidly and accurately identify the gender of Yellow River C. carpio. Furthermore, the sex-specific chromosome region may be characterized and used to study mechanisms of chromosome evolution in this fish species.     

Molecular detection and quantification of the protozoan Bonamia ostreae in the flat oyster, Ostrea edulis

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR^® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of <1% and a minimum detection limit of 50 gene copies per reaction. Using infected oyster samples as templates, the assay was at least 10-fold more sensitive than conventional PCR. The quantitative assay was applied to test 132 oysters, and results were compared with the heart imprint method. There was a strong correlation between both techniques, and the results showed that the real-time PCR assay should be useful for studies of the ecology of B. ostreae and its host–parasite relationship.     

Polymerase chain reaction for the detection ofPseudomonas aeruginosa,Stenotrophomonas maltophiliaandBurkholderia cepaciain sputum of patients with cystic fibrosis

Occurrence ofPseudomonas aeruginosa,Stenotrophomonas (Xanthomonas) maltophiliaandBurkholderia (Pseudomonas) cepaciain sputum of cystic fibrosis (CF) patients was demonstrated with a simple and rapid polymerase chain reaction (PCR) technique. The PCR was performed with a set of three primer pairs based on 16S rRNA sequences after sputum preparation with dithiothreitol and NaOH lysis. All three pathogens could be individually detected by the use of this technique. To prevent carry-over contamination, dUTP and uracil-N-glycosylase were included in the reaction. The amplicons were visualized by agarose gel electrophoresis. Sputum culture was performed on all samples. Ninety specimens from CF patients were analysed. The sensitivity for the detection ofP. aeruginosawas 37/40 (93%) compared to culture. Bacterial growth ofP. aeruginosawas found in three cases, where PCR amplicons were not detected, while PCR was positive in five cases, where culture did not reveal the presence of this bacterium. For this reason, the specificity was 45/50 (90%). ForS. maltophilia, the PCR was less sensitive than culture (positive in three of six cases). In our series,B. cepaciawas detected by culture in one case and this was also detected by PCR. There were no false-positive PCR results regardingS. maltophiliaorB. cepacia. Thus, combined PCR-based detection of these three clinically relevant bacteria in sputum samples from CF patients can be performed by a reliable technique in a relatively simple manner. The present data indicate a high sensitivity and specificity forP. aeruginosa. The lower sensitivity observed for the detection ofS. maltophiliain sputum andB. cepacia, as estimated from laboratory strains, may depend on PCR conditions and genetic heterogeneity, respectively. The greatest gains with this method can be made when it is used for the early detection ofP. aeruginosain sputum-producing CF patients.     

Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus

Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.     

Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay

A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344(T). B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.     

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

The nucleotide sequence of ac936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal speciesStachybotrys chartarumand for other species ofStachybotrysand the related genusMemnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers forS. chartarumin the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo® 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® 5.0 software. The automated computer searches were unsuccessful in findingS. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that includedS. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for onlyS. chartarumand one other species ofStachybotrys. 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 forS. chartarum. The relative merits of different PCR strategies for the detection ofS. chartarumemploying either one or both of the primers identified in this study are discussed.     

Differentiation of seventeen genospecies ofAcinetobacterby multiplex polymerase chain reaction and restriction fragment length polymorphism analysis

The 17 described genomic species (DNA groups) of the genusAcinetobacter, including the type strains of the seven named species, were studied by using a multiplex PCR. The multiplex PCR assay combined two primer sets (rA1 and rA2 forrecAgene target; rib1 and rib2 for 16S rDNA sequence) in a single reaction. Restriction analysis with two enzymes (MboI andHinfI) of the enzymatically amplified products allowed identification of all genospecies. This technique proved to be a rapid and reliable method for the identification of theAcinetobactergenomic species, including the closely related DNA groups (1, 2, 3, 13). The results of this study suggest that the proposed method can be used for the identification ofAcinetobacterspp. and as such may help to elucidate the ecology and clinical significance of the different species of this genus.     

Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS 900 -like sequences detectable by IS 900 polymerase chain reaction: implications for diagnosis

PCR targeting the 5′ end of IS 900 has been considered specific for identification ofMycobacterium avium subsp. paratuberculosis and is frequently applied to confirm the presence of this organism in the diagnosis of Johne's disease. IS 900 PCR has also been applied to studies of the aetiology of Crohn's disease. Mycobacterium spp. isolated from the faeces of 3 clinically normal animals in 2 Australian states appeared not to be M. paratuberculosis but were positive by IS 900 PCR. The isolates were characterized using mycobactin dependency, biochemical tests, IS 900 and 16 S rRNA sequencing and restriction fragment length polymorphism (RFLP) using IS 900 as probe. DNA sequencing confirmed that the isolates had between 71% and 79% homology with M. paratuberculosis in the region of IS 900 amplified, were most closely related to Mycobacterium scrofulaceum, and confirmed the usefulness of restriction enzyme analysis of amplified product to identify the false positive results. RFLP analysis with Bst Ell detected three to five copies of the IS 900 -like element in the isolates. These were located in molecular weight fragments that were clearly different to IS 900 in previously characterized strains of M. paratuberculosis. It is likely that these isolates are environmental mycobacteria. Southern blotting with an internal probe is unlikely to provide differentiation of M. paratuberculosis from these organisms. We recommend the adoption of restriction endonuclease analysis of IS 900 PCR product as a routine precaution to prevent the reporting of false positive IS900 PCR results.