Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S / Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG). La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5por ciento fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG(AU)

OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU). Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5percent were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using(AU)

[Development of a multiple PCR method for the identification of Ureaplasma parvum and Ureaplasma urealyticum]. / Desarrollo de un método de PCR-múltiple para la identificación de Ureaplasma parvum y Ureaplasma urealyticum.

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the "MYCOFAST Evolution-2" Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70% were U. parvum and 30% U. urealyticum; in 76 positive samples in women, 83% corresponded to U. parvum and 17% to U. urealyticum, while in 63 positive samples of men, 76% identified U. parvum and 24% U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

Desarrollo de un método de PCR-múltiple para la identificación de Ureaplasma parvum y Ureaplasma urealyticum / [Development of a multiple PCR method for the identification of Ureaplasma parvum and Ureaplasma urealyticum]

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the [quot ]MYCOFAST Evolution-2[quot ] Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70

were U. parvum and 30

U. urealyticum; in 76 positive samples in women, 83

corresponded to U. parvum and 17

to U. urealyticum, while in 63 positive samples of men, 76

identified U. parvum and 24

U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

Desarrollo de un método de PCR-múltiple para la identificación de Ureaplasma parvum y Ureaplasma urealyticum. / [Development of a multiple PCR method for the identification of Ureaplasma parvum and Ureaplasma urealyticum]

Ureaplasma parvum and Ureaplasma urealyticum, also known as biovar parvum and biovar T960, respectively, could be associated with several disorders in men, women, and mainly, in newborn children with under weight. Several methods have been developed in order to identify the species or biovars of ureaplasmas. We developed a Multiplex-PCR method using the UPS-UPSA and UUS2-UUA2 primers, specific for U. parvum and U. urealyticum, respectively. This Multiplex-PCR method was used to identify cultures of clinical positive samples to Ureaplasma spp. by the [quot ]MYCOFAST Evolution-2[quot ] Kit. Of 56 positive cultures to Ureaplasma spp. from newborn children, 70

were U. parvum and 30

U. urealyticum; in 76 positive samples in women, 83

corresponded to U. parvum and 17

to U. urealyticum, while in 63 positive samples of men, 76

identified U. parvum and 24

U. urealyticum. The PCR-multiplex method showed specificity for the identification of the biovars or species of ureaplasmas of clinical interest.

In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR.

Mycoplasma bovis and Mycoplasma agalactiae are two very closely related species which cause mastitis in cows and goats, respectively. M. bovis can also cause arthritis and respiratory disease in cattle. It has recently been shown that the 16S rRNA sequences differ only in 8 nucleotide positions between the two species [J.G. Mattsson, B. Guss and K.-E. Johansson (1994) FEMS Microbiol. Lett., 115: 325-328]. These nucleotide differences are distributed over the molecule in such a way that it is difficult to design specific identification systems, based on PCR only, for M. bovis and M. agalactiae. Two different PCR systems based on 16S rRNA sequence data have, however, been designed for these two species. The forward primers were identical in the two systems and complementary to a segment of the evolutionarily variable region V2. The reverse primers were complementary to the variable region V6, in which there are two nucleotide differences between M. bovis and M. agalactiae. The size of the PCR products, generated with these primers, was 360 bp. Cross-amplification was obtained with the two species in the heterologous PCR systems, but with approximately a 100-fold lower efficiency. Cross-amplification was not obtained with any other bovine or caprine mycoplasma except for Mycoplasma sp. strain A1343 of the caprine group 7. The detection limit of the PCR system for M. bovis with a reference culture was 4 x 10(2) CFU/ml and of the PCR system for M. agalactiae 2 x 10(2) CFU/ml. The M. bovis-PCR system was used to analyze nasal samples of calves from a herd where an outbreak of pneumonia had occured and it proved possible to detect M. bovis in these samples.