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PubMedDate not supplied

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR.

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Biomarkers↗
PubMed2004

"Multiplex PCR" identification of the atypical and monophasic Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- in São Paulo State, Brazil: frequency and antibiotic resistance patterns.

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.

Animals↗
PubMedDate not supplied

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing.

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Adult↗
PubMed2003

An assessment of the Roche Amplicor Chlamydia trachomatis/Neisseria gonorrhoeae multiplex PCR assay in routine diagnostic use on a variety of specimen types.

The Roche Cobas Amplicor Chlamydia trachomatis/Neisseria gonorrhoeae polymerase chain reaction (PCR) assay can simultaneously detect both C. trachomatis and N. gonorrhoeae, and has been cleared by United States Food and Drug Administration (FDA) for the testing of endocervical and urethral swabs and urine specimens. The Amplicor N. gonorrhoeae PCR target sequence is known to be present in some strains of commensal Neisseria species, including N. cinerea and N. subflava, necessitating the use of a second PCR assay to confirm positive results. This study analyses the performance of the assay on 7,007 unselected specimens submitted to the laboratory for the PCR diagnosis of N. gonorrhoeae and C. trachomatis; compares the PCR assay with culture for the detection of N. gonorrhoeae; examines the performance of the assay with specimens from different body sites; and briefly compares two confirmatory PCR assays. Confirmation rates for an initial Amplicor N. gonorrhoeae positive result varied widely by specimen type, ranging from 86.2 per cent for penile/urethral swabs to 5.6 per cent for oropharyngeal swabs, indicating all positive Amplicor N. gonorrhoeae results should be confirmed by a second method to maintain adequate specificity. Overall there was 98.1 per cent agreement between the confirmed PCR assay and culture, with confirmed PCR showing a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 81.7 per cent, 99.5 per cent, 92.7 per cent and 98.5 per cent respectively, compared with N. gonorrhoeae culture. When confirmed C. trachomatis/N. gonorrhoeae PCR assay performance was analysed against culture using only FDA-cleared specimens (553 penile/ urethral swabs, urines and cervical/vaginal swabs), sensitivity, specificity, PPV and NPV and percent agreement were 96.7 per cent, 99.8 per cent, 98.9 per cent, 99.4 per cent and 99.3 per cent respectively. No significant differences were found between the two confirmatory PCR assays used during the study period. Limitations of Amplicor for the detection of N. gonorrhoeae and the appropriate use of combined C. trachomatis/N. gonorrhoeae PCR in a routine diagnostic setting are discussed.

Cervix Uteri↗
PubMed2001

DNA testing of Klinefelter's syndrome in a criminal case using XY chromosomal STR multiplex-PCR.

We report genetic typing of Klinefelter's syndrome applied to casework in forensic DNA testing. In this case, by using extracted DNA from body samples (muscle and bones), we could identify two distinct X alleles in two out of three X-STR loci (HPRTB and ARA), in addition to Y alleles (DYS390, DYS393). The extra X was found to have originated from father, and the victim turned out to have 47XXY Klinefelter's syndrome. The victim was a 30-year-old male, born from relatively elderly parents as a second child. His father was a severe alcoholic and had been malnourished for more than 20 years at the moment of his birth. He exhibited slight mental retardation as a child, and belonged to a criminal group as an adult. The method presented here was useful to accurately diagnose sex chromosomal abnormality instead of conventional chromosomal analysis and Xg blood group typing. A subtype of this syndrome, 48 XXXY or mosaic, for example, could be identified if the intensity of the overlapped X bands were calculated.

Adult↗