Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples.

We have used the polymerase chain reaction to detect Mycoplasma genitalium in artificially seeded human throat swab samples as well as in clinical material. On the basis of the published nucleotide sequence of the M. genitalium 140-kDa adhesin gene, primers were chosen to produce an amplified fragment of 281 bp. Five different previously isolated strains, including the type strain of M. genitalium, could all be detected by the polymerase chain reaction, and DNAs from other mycoplasmal and bacterial species yielded negative results. The detection limits were estimated to be approximately 50 organisms by inspection of ethidium bromide-stained agarose gels and 4 organisms when a biotinylated oligonucleotide probe was used in filter hybridization. The amplified DNA fragments were subjected to restriction enzyme analysis. DNAs from the five different isolates all possessed EcoRI, SspI, AluI, Sau3AI, and DdeI restriction sites, as predicted from the published sequence. A total of 150 urogenital swabs collected from 100 patients for culturing of Chlamydia trachomatis were tested for the presence of M. genitalium DNA. Ten samples from eight patients were found positive. The amplified DNA fragments from all of our clinical samples possessed the AluI, Sau3AI, and DdeI restriction sites, but three samples from two patients did not contain the SspI site and none of the samples contained the EcoRI site.     

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.     

Improved detection of Shigella using Escherichia coli medium enrichment: Polymerase chain reaction from stool samples

Laboratory diagnosis of shigellosis using conventional culture technique is limited by lower sensitivity and higher turnaround time. Here, we have evaluated the role of polymerase chain reaction from stool samples after enrichment in Escherichia coli medium for detection of Shigellae. The technique not only increased the sensitivity but also decreased the turnaround time.     

Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples.

AIMS: To evaluate the use of a cppB gene derived polymerase chain reaction (PCR) assay for direct detection of Neisseria gonorrhoeae in clinical samples. METHODS: A PCR assay was performed on 33 N gonorrhoeae strains and 12 other Neisseria species and other normal genital flora to evaluate the specificity of the chosen cppB primers. The assay was subsequently evaluated with 52 clinical swab samples collected from China. RESULTS: An amplified product of 390 base pairs (bp) was observed with all the N gonorrhoeae strains, each of these products on digestion with the restriction enzyme MspI produced two bands of 250 bp and 140 bp respectively. This set of primers did not produce any amplified product of the expected length with the other non-gonococcal strains tested. For the 52 clinical swabs, 34 were culture positive and PCR successfully detected all these positives. In addition the PCR was positive for two swabs which were culture negative but positive for N gonorrhoeae antigens when tested with the ELISA method (Gonozyme). CONCLUSIONS: This PCR assay is a promising diagnostic tool for detection of gonococci directly from clinical swab samples. Further evaluation is necessary.     

Polymerase chain reaction for detection of Mycoplasma gallisepticum in environmental samples

The polymerase chain reaction (PCR) was used to detect Mycoplasma gallisepticum in samples collected from the environment of experimentally or naturally infected poultry. Culture was also used in the experimental infections. Of 160 samples of food, drinking water, feathers, droppings or dust collected during experimental infection, 103 were positive using a M. gallisepticum -specific PCR (MG-PCR) and 68 were positive using a PCR (mycoplasma-PCR) that detects all species of the genera Mycoplasma , Spiroplasma, Acholeplasma and Ureaplasma . Six of these samples were also positive by culture. In environmental samples collected on a depopulated M. gallisepticum -positive turkey farm, three and two out of a total of 12 were positive by mycoplasma-PCR and MG-PCR, respectively. These results indicate the disseminating capacity of this mycoplasma and the possible use of PCR methods for epidemiological analyses and control of farm decontamination before the introduction of new birds.     

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.     

Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses

A polymerase chain reaction combined with restriction enzyme analysis was developed for detection and differentiation of all 12 fowl adenovirus (FAdV) serotypes representing the five fowl adenovirus (A to E) species. For primer design, the published sequences of the hexon proteins of FAdV1, FAdV8 and FAdV9 were aligned and conserved regions in the two pedestal regions adjacent to the L1 loop region were determined. A primer pair (hexon A/hexon B) was constructed and was shown to amplify approximately 900 bp of the hexon gene including the L1 loop region. An amplification product was detected using supernatant of infected cell cultures from all FAdV1 to FAdV12 reference strains used in our study. The sequence and the restriction patterns of the hexon A/B fragments of the 12 FAdV strains were determined and compared. The successive use of four different endonucleases allowed the complete differentiation of the reference FAdV strains. Twenty-six fowl adenoviruses isolated during our routine virological diagnosis activities could all be amplified using hexon A/hexon B primers. Restriction analysis results showed that 8/26 adenovirus strains contained two different FAdV types. FAdV4, FAdV12, FAdV1, FAdV5 and FAdV6 were the most frequently isolated.     

Polymerase chain reaction for detection of Mycobacterium tuberculosis.

A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis.     

Polymerase chain reaction assay for detection of human cytomegalovirus.

Direct detection of human cytomegalovirus (HCMV) from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying HCMV DNA. The efficiency of the amplification reaction was examined by using three different buffers and concentrations of deoxynucleotide triphosphates. The PCR assay was most efficient with a reaction mixture containing 17 mM ammonium sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each deoxynucleotide triphosphate at a final concentration of 1.5 mM. After 35 cycles of amplification, 0.15 fg of a plasmid containing the cloned target gene (corresponding to approximately six gene copies) was detected. The PCR assay correctly identified all of 24 clinical isolates of HCMV. Virus in urine specimens could be disrupted by heating at 93 degrees C for 30 min. The viral DNA was amplified directly from 5 microliters of preheated urine, with no further treatment before amplification. We tested the PCR assay on urine specimens from patients who had undergone renal transplantation that had been screened for the presence of HCMV by enzyme-linked immunosorbent assay, hybridization assay, and direct virus isolation. Specimens that were positive by one or more of these assays were screened by PCR. HCMV was consistently detected by PCR in all specimens that were positive by at least one other test. No cross-reactivity to other herpesviruses or MRC-5 cellular DNA was observed.     

Polymerase chain reaction for detection of Toxoplasma gondii

A DNA-based assay has been developed for the detection of Toxoplasma gondii. The assay makes use of the polymerase chain reaction (PCR) to amplify part of the P30 gene on the parasite's DNA. Following gel electrophoresis, the amplified DNA can be detected either directly on the gel or by Southern hybridisation with radioactive or non-radioactive DNA probes. The assay has been used to detect the DNA from different isolates of T. gondii in a background of human or mouse DNA. Together with other information such as clinical data, CT scans and serology, the PCR assay should improve the diagnosis of toxoplasmosis in immunosuppressed and immunocompromised patients as well as in fetal tissues.     

Polymerase chain reaction for detection ofMycobacterium tuberculosis in sputum

The polymerase chain reaction (PCR) was evaluated in a trial which, with respect to the positive-to-negative ratio, approximated the situation of a diagnostic laboratory in a tuberculosis-endemic area. Three hundred sputum samples were included in the study, of which one-third were known to contain mycobacteria as judged by direct microscopy. The repetitive insertion sequence IS6110/IS986 ofMycobacterium tuberculosis was used as a target. The samples were spiked with DNA from a modified IS6110/IS986 sequence, which gives rise to PCR products easily distinguished from PCR products amplified from chromosomalMycobacterium tuberculosis DNA. This allowed identification of samples that contained substances inhibitory to theTaq polymerase. The detection limit of the assay was 0.05 pg to 0.5 pg of purifiedMycobacterium tuberculosis DNA, corresponding to 10 to 100 organisms. The sensitivity and specificity of the PCR was compared with that of conventional microscopy and culture. It was concluded that this method is fast and sensitive, but that culture currently is crucial for assessing viability and thus infectivity.     

Polymerase chain reaction for detection of herpes simplex virus encephalitis.

A patient with herpes simplex virus encephalitis is described. The polymerase chain reaction (PCR) was used to confirm the diagnosis in cerebrospinal fluid. PCR allows the rapid diagnosis of many infectious organisms, such as HSV, in which prompt diagnosis is essential.     

Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens.

The polymerase chain reaction based on the selective amplification of a 531-bp fragment of the gene encoding the proline-rich antigen of Mycobacterium leprae was applied to nasal swab specimens from leprosy patients, occupational contacts, and endemic and nonendemic controls. To prevent false-positive amplification, we used dUTP and uracil-DNA-glycosylase in all polymerase chain reactions. False-negative reactions were detected by using a 531-bp modified template as an internal control. Amplification products were found in 55% of untreated patients, in 19% of the occupational contacts, in 12% of endemic controls, and in none of the nonendemic controls. This study strongly suggests that not only leprosy patients but also healthy persons may carry M. leprae. We concluded that polymerase chain reaction is a reliable method to detect M. leprae in nasal specimens. The method holds promise for studying the spread and transmission of M. leprae within a population.     

Polymerase chain reaction for rapid detection of ocular adenovirus infection

Adenoviruses are associated with endemic and epidemic acute conjunctivitis, large nosocomial outbreaks reflecting virus transmission on unwashed hands or inadequately sterilised ophthalmic instruments. The polymerase chain reaction (PCR) proved more sensitive than antigen detection by immune dot-blot test for the rapid diagnosis of ocular adenovirus infection (sensitivities in a retrospective study 112/123 (91%) versus 72/123 (59%), P< 0.001). Indeed, in a prospective comparison, DNA amplification and virus isolation generated similar numbers of positive results (34 versus 32), though five PCR positive results were possibly false positives. The sensitivity of the PCR was largely independent of adenovirus subgenus or serotype, though reduced sensitivity with subgenus B strains could not be excluded. Specimen preparation for DNA amplification using a simple lysis buffer proved more effective than phenol-chloroform extraction. The immune dot-blot test gave unavoidable false positive results, but with the PCR this problem could be minimized by technical modifications. The PCR could replace antigen detection and virus isolation as the initial test for adenoviruses in conjunctival swabs, with cell culture only being retained for adenovirus serotyping in PCR positive specimens and for other viruses such as herpes simplex. © 1995 Wiley-Liss, Inc.     

Polymerase chain reaction detection of foodborne Salmonella spp. in animal feeds

Foodborne salmonellosis continues to be a public health issue of considerable concern. Animal feed has been a major link in pre-harvest food animal production. Although monitoring systems and control measures are available to limit Salmonella spp. contamination on animal feeds detection methodology is relatively time consuming in the context of time inputs for feed processing and mixing. Current cultural methods of Salmonella spp. detection in feeds require several days for confirmation. This amount of time represents significant problems if control measures are to be effectively implemented in a fashion that keeps feed processing costs low. Molecular methods offer improved sensitivity and potential reduction in assay time. In particular, several commercial polymerase chain reaction (PCR) assays, and combined PCR-hybridization assays have been suggested as possible means to implement more rapid detection of Salmonella spp. extracted from animal feeds. It has now become possible to rapidly detect and confirm the presence of foodborne Salmonella spp. in feed matrices by commercial amplification detection systems. The primary challenges remaining are to develop more reliable recovery and extraction procedures for routine processing of samples from a wide variety of feed matrices and apply molecular techniques for assessing physiological status of Salmonella spp. contaminants in animal feeds.