Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay.

Rapid detection of toxigenic Clostridium difficile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples.     

Nested polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

The nested polymerase chain reaction technique was compared with the conventional smear and culture methods for detection of Mycobacterium tuberculosis. The nested polymerase chain reaction used in this study showed excellent specificity, sensitivity, and agreement with the conventional methods for 417 clinical samples, indicating a contribution to the rapid diagnosis of mycobacterial infectious diseases.     

Real-time polymerase chain reaction assay for detection of Streptococcus pneumoniae in sputum samples from patients with community-acquired pneumonia.

BACKGROUND AND PURPOSE: A quantitative real-time polymerase chain reaction (PCR) method targeting the pneumolysin gene of Streptococcus pneumoniae in sputum specimens from patients with community-acquired pneumonia (CAP) was evaluated for the identification of pneumococci. METHODS: The applicability of the assay to clinical samples was evaluated by studying 140 sputum specimens from patients with CAP. Of the specimens, 96 (68.6%) were found to be positive by real-time PCR. The results were compared to culture findings. RESULTS: The sensitivity and specificity of the real-time PCR assay developed in this study as compared to those of the culture method were 97.2% and 60.9%, respectively. CONCLUSION: Real-time PCR assay was found to be a rapid and sensitive method for the detection of pneumococci.     

Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples.

A method based on nested polymerase chain reaction was developed for the detection of enteroviral genomes in biological samples. By taking advantage of the conserved 5' noncoding region of the enteroviral RNA, two sets of primers were utilized, enabling the detection either of a broad range of enteroviruses or of group B coxsackieviruses only. The sensitivity of the method is close to the detection of single molecules of viral RNA in as much as 1 mg of tissue sample. A preliminary study showed the usefulness of this technique for the analysis of endomyocardial biopsy samples from patients with idiopathic dilated cardiomyopathy and myocarditis.     

Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples

Objective: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S–1-specific probe.
Methods: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella bumetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method.
Results: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S–1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test.
Conclusions: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.     

Multiplex polymerase chain reaction for subgenus-specific detection of human adenoviruses in clinical samples

The polymerase chain reaction (PCR) has been used previously for the detection and typing of adenoviruses directly in clinical samples. Since under clinical conditions subgenus-specific identification is often sufficient, we extended the genus- and type-specific PCR by a subgenus-specific PCR. By sequencing several loop I4 gene regions of the hexon (major adenovirus coat protein) and comparing them to published sequences, subgenus-specific sequences were identified in this region. By using primers targeted to this region and to a conserved hexon gene region, a multiplex, nonnested PCR for the detection and subgenus-specific identification of adenoviruses could be established. The six subgenus-specific amplimers are distinguishable by agarose gel electrophoresis, and subsequent restriction analysis is not necessary. The specificity of the subgenus-specific primer pairs was tested on 23 adenovirus prototypes, representing all six subgenera, on 9 subgenus B and D intermediate strains, and on 16 subgenus C genome types. Furthermore, multiplex, subgenus-specific PCR was performed directly with 100 clinical specimens, including stool samples, ocular swabs, and throat swabs. Adenoviruses of all subgenera could be detected. Especially for clinical application, the rapid, one-step differentiation between subgenus D adenoviruses, causing the severe and highly contagious epidemic keratoconjunctivitis, and subgenus B and E adenoviruses, causing relative harmless ocular infections, is of great importance. The subgenus-specific PCR could also facilitate the primary classification of unknown virus isolates.     

The polymerase chain reaction for Mycoplasma gallisepticum detection

On the basis of the aligned 16S rRNA sequences of Mollicutes, a pair of primers was chosen for the detection of Mycoplasma gallisepticum. When used in the polymerase chain reaction (PCR), the primers detected a specific amplification of all Mg strains tested, yielding an expected 330 bp product. Amplification was not detected when other Mollicutes or E. coli were used as PCR templates. SPF chickens were experimentally inoculated with two strains of M. gallisepticum or Mycoplasma iowae. Tracheal swabs were collected 8, 15, 20 and 28 days after inoculation, and cultured for mycoplasma or tested by PCR. PCR products were detected by hybridization with a digoxigenin-labeled probe and by chemilumines-cence. The results showed that culture was positive for 49/73 swabs while PCR detected 70/72 positive samples. Thus, PCR can provide the basis of a sensitive, specific, rapid and non-radio-active method for detecting M. gallisepticum.     

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples

Purpose: To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods: A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results: Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion: Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).     

Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

AIM--To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS--Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS--The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION--Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory.     

Detection of Borrelia burgdorferi in biological samples using the polymerase chain reaction assay

Oligonucleotide primers were used in the polymerase chain reaction assay to amplify specific DNA regions of the Borrelia burgdorferi 49-kb linear plasmid. One set of primers identifies a 442-bp DNA fragment in the OspA gene and a second pair of amplimers, a 176-bp DNA piece located in the OspB gene. The last set of primers, OspBpc3/pc4, outperformed the other pair in discriminating pathogenic North American or European isolates from related bacterial species, detected down to 4 spirochaetes, and was suitable for the identification of B. burgdorferi in biological samples, such as synovial and cerebrospinal fluids.     

Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples.

In order to improve the diagnosis of Listeria meningitis or meningoencephalitis, especially in patients who have received antibiotics before their cerebrospinal fluid (CSF) has been examined, two assays for the detection of Listeria monocytogenes based on the polymerase chain reaction (PCR) were evaluated. After a standard PCR, the amplified DNA was detected either by a second round of PCR with internal primers followed by gel electrophoresis and ethidium bromide staining (nested PCR) or by dot blot hybridization to an internal digoxigenin-labeled probe (PCR-dot blot). For PCR, two sets of primers within the invasion-associated protein gene (iap gene) were chosen. They allowed for the highly specific detection of all L. monocytogenes reference strains tested (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 7). These primers did not detect amplification products from various other gram-positive or gram-negative bacterial DNAs or human DNA. The sensitivities of both assays were assessed on sterile CSF samples that were artificially seeded with serial dilutions of L. monocytogenes serotype 4b cells. By both methods the limit of detection was less than 10 cells in the initial reaction. Since the nested PCR is more prone to contamination because of manipulation of the amplified products, a standard PCR assay followed by dot blot hybridization was applied to 52 CSF samples in a retrospective study. Of 28 CSF samples which were sterile or positive for bacteria other than Listeria species, 24 were PCR negative. In contrast, from 17 patients with culture-proven Listeria meningitis, 14 of 17 initial CSF samples were PCR positive, as were 3 of 7 culture-negative followup CSF samples taken after patients received antibiotics. These results support the usefulness of this approach in the diagnosis of Listeria meningitis, in particular, when antibiotic administration precedes culture of CSF.     

The polymerase chain reaction for detection of T-cell antigen receptor expression.

Applications of the polymerase chain reaction have revolutionized the field of immunogenetics, particularly in studies of human leukocyte antigen class II polymorphism, and more recently in the analysis of T-cell receptor usage. However, the enormous diversity and variability of the T-cell receptor complex have made the amplification of the complete repertoire difficult. Several methods have been devised to address this problem. Each system is described with recent examples of its use and an assessment of its advantages and disadvantages. The use of quantitative polymerase chain reaction in T-cell receptor analysis is also discussed. The elucidation of the T-cell repertoire involved in a pathogenic process can have therapeutic implications, given the success of reversing experimental autoimmune disorders by directing specific forms of immunotherapy against V region gene products.     

Specific and sensitive two-step polymerase chain reaction assay for the detection ofSalmonella species

A polymerase chain reaction (PCR) assay was applied for the selective amplification of a characteristic sequence within aSalmonella-specific chromosomal fragment. A two-temperature PCR cycle enhanced both the speed and overall sensitivity of the amplification procedure. Twenty-one well-characterizedSalmonella strains and a number of non-Salmonella strains were tested. With the exception of the rarely isolatedSalmonella arizonae strain, the PCR-based approach enabled the specific identification ofSalmonella with a detection limit of 10³ organisms. In combination with a nested PCR assay, as few as ten organisms were detectable. Specificity was demonstrated as no distinct amplification products were detectable with any of the testednon-Salmonella strains. With a pre-enrichment step using paramagnetic antiSalmonella beads, an increase in sensitivity was observed in the case of clinical samples while the amplification process was not influenced.     

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orbivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 x 10(3) TCID50 of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID50/ml). However, at 28 days p.i. (less than 1.6 TCID50/ml) BTV RNA was not detected using the PCR in either sample.     

Evaluation of an in-house polymerase chain reaction for detection of Neisseria gonorrhoeae in urogenital samples

AIM: To develop and evaluate a one day in-house polymerase chain reaction (PCR) assay for the detection of Neisseria gonorrhoeae DNA in urogenital samples. METHODS: 429 urogenital specimens were tested for the presence of N gonorrhoeae by in-house PCR and by Gen-Probe. The PCR assay amplifies target sequences within the N gonorrhoeae cppB gene on the 4.2 kb cryptic plasmid, after which amplicons are detected by a streptavidinbiotin based enzyme immunoassay using an internal probe. Discordant specimens were further evaluated by repeating the PCR and the Gen-Probe assay, and by an additional PCR using another set of 16S primers followed by radioactive detection of amplicons on a Southern blot. RESULTS: Of the 429 samples tested, 15 were found positive by in-house PCR, eight of which were confirmed by Gen-Probe. Of the seven discrepant samples, five were confirmed by 16S PCR and are also considered true positive. The remaining two samples were positive in the in-house PCR only, and are considered false positive. After resolution of discrepant samples, the sensitivities of the N gonorrhoeae assays were 100% and 61.5% for the in-house PCR and Gen-Probe, respectively, while specificities were comparable at 99.5% and 100%. CONCLUSIONS: The in-house PCR for the detection of N gonorrhoeae DNA is at least comparable to Gen-Probe in performance. An extended evaluation period should elucidate if the additional five GO-PCR positive specimens, confirmed by 16S PCR, are caused by persistence of DNA or whether the method is indeed more sensitive.     

Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction.

A single pair of oligonucleotide primers was used for polymerase chain reaction amplification of a 212- or 215-bp region of Escherichia coli Shiga-like toxin (SLT) genes from crude fecal culture extracts. Genes were typed by hybridization of the polymerase chain reaction products to SLT-I- or SLT-II-specific oligonucleotide probes. The procedure was capable of detecting fewer than 10 SLT-producing E. coli organisms per ml of culture against a background of more than 10(9) other organisms per ml and provides a rapid and sensitive means of screening primary fecal cultures for the presence of such strains. When this procedure was used to test primary cultures from gut contents or feces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT-II sequences.     

Human papillomavirus detection in urine samples from male patients by the polymerase chain reaction.

Human papillomavirus (HPV) detection was performed using the polymerase chain reaction technique on urine samples from 17 male patients with condylomata acuminata in the meatus urethrae. Urine samples from 14 male laboratory volunteers were analyzed as controls. The DNA was extracted and purified from urine sediments, centrifuged at 1,800 and 100,000 x g, and subjected to 40 cycles of amplification with HPV 6 and HPV 11 type-specific anticontamination primers and the heat-stable Taq DNA polymerase. HPV was detected in the urine of 15 (88%) patients. In all positive patients the urine sediments of both the 1,800 and 100,000 x g centrifugation steps contained HPV DNA. Eight patients were found to be positive for HPV 6 DNA, six were positive for HPV 11 DNA, and one was positive for both HPV 6 and HPV 11 DNA. None of the males in the control group was positive for either HPV 6 or HPV 11 DNA. The results demonstrate that HPV can be transported by the urine, probably in exfoliated HPV-infected cells. A similar mechanism may occur during ejaculation, allowing sexual transmission of HPV viruses harbored in the cells of the male genital tract.     

Polymerase chain reaction for detection of adenoviruses in stool samples.

The usefulness of the polymerase chain reaction (PCR) method for diagnosing adenovirus infections was investigated. Several primers, including primers specific for the hexon-coding region and for enteric adenovirus types 40 and 41, were evaluated. The PCR method was validated against cell culturing in routine diagnostic work and against restriction enzyme analysis of viral DNA. Sixty diagnostic specimens were selected for evaluation by the PCR method. Twenty of the 60 specimens were found positive on the basis of cytopathic effects and latex agglutination (Adenolex [Orion Diagnostica, Helsinki, Finland]), and 16 were identified and typed as adenoviruses by polyacrylamide gel electrophoresis. PCR was performed on all 60 specimens in parallel directly on diluted stool samples and on viral DNA extracted from cells inoculated with the same stool samples. When the general hexon primers were used 51 of the 60 specimens from infected cell cultures were found positive by PCR, whereas only 13 specimens were found positive when PCR was performed directly on stool samples. With the use of selective primers for enteric adenoviruses 16 of the 60 cell cultures were found to exhibit amplification products by PCR, whereas 4 were detected in stool samples. None of the 60 specimens were found positive by PCR when an adenovirus type 40-specific primer pair was used. PCR was found to be a fast, sensitive, and reliable method for the detection of adenoviruses in diarrheal disease, provided the amplifications were performed directly on diluted stool samples.