Molecular Detection Methods

Introduction of molecular detection techniques provides a new horizon in the identification anddetection of Mycoplasma and Ureaplasma species. These provide improved specificity and sensitivity compared with traditional culture methods. The molecular detection techniques on Mycoplasma and Ureaplasma include probe hybridization and PCR methods.

Probe Hybridization

Sequences based on the variable species-specific regions of 16S rRNA genes have been widely used for synthesis of specific probes for hybridization. Several rRNA probe hybridization techniques have been developed for detecting mycoplasmas that are of human origin, although many rRNA probes have been designed for the purpose of contamination detection in tissue cell cultures (Razin, 1994; Kessler et al., 1997). Specific probes that are designed from genomic libraries of Mycoplasma, M. pneumoniae and M. genitalium, have also been reported (Hyman et al., 1987). Dot blot hybridization has been variously designed in these probe methods with 32P-labeled, digoxigenin, or biotin-labeled probe for detection of Mycoplasma. A

major limitation has been that the detection limit of these assays is approximately 1 ng of specific mycoplasma DNA or 104-105 CFU, which is often not sufficiently sensitive for use in the clinical laboratory (Marmion et al., 1993; Razin, 1994). Recently, a rapid detection method for M. genitalium, M. hominis, U. parvum, andU. urealyticum in genitourinary samples by PCR-microtiter plate hybridization has been developed (Yoshida et al., 2003). In this assay, four species-specific capture probes were used to detect the targets by PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization. Sensitivity of this assay was up to detection of approximately 10 copies of the 16S rRNA gene of each of the four species. Further evaluation may therefore prove this to be a useful tool for diagnosis of genitourinary infections with Mycoplasma or Ureaplasma.

Compared with what was available a decade ago, many PCR systems have been developed for detection of clinically relevant Mycoplasma and Ureaplasma (Blanchard et al., 1993; Bebear et al., 1997; Yoshida et al., 2002). Specific primers have been designed for different target regions including 16S rRNA genes and other repetitive sequences, such as MgPa adhesion gene of M. genitalium (Jensen et al., 1991; Baseman et al., 2004). In particular, Ureaplasma species were detectable by specific PCR amplification on urease genes (Blanchard et al., 1993). As PCR assay is highly sensitive compared with culture, it is a potentially useful tool for detection of slow-growing and fastidious organisms, such as M. genitalium, where only very small amount of bacteria DNA is required. M. genitalium is also the smallest known self-replicating cell with a genome size of 580 kb only. This imposes severe biosynthetic limitations. The highly fastidious nature of M. genitalium is also reflected in the absence of direct isolations from urethral specimens (Tully et al., 1981). With the many difficulties in cultivating M. genitalium, PCR assay has become an important tool to establish links between M. genitalium and human diseases. Two different TaqMan-based real-time PCR assays for quantifying M. genitalium were recently developed and have been validated in detection of a fragment of MgPa adhesion gene or 16S rRNA (Jensen et al., 2004; Yoshida et al., 2002). These TaqMan assays can provide valuable information for understanding the pathogenic mechanisms of M. genitalium in causing urogenital tract disease.

Apparently, PCR assays seem to be less valuable in other more rapidly growing and easily cultivable Mycoplasma, such as M. hominis, and Ureaplasma species. U. urealyticum can often be isolated with ease, although it may be difficult to culture from certain specimens such as amniotic fluids and endotracheal aspirates of newborns (Blanchard et al., 1993). The use of PCR technology in these situations, in contrast to culture, can also enhance the detection of other mycoplasmas. A multiplex PCR assay has been developed to detect C. trachomatis, N. gonor-rhoeae, U. urealyticum, and M. genitalium in first-void urine specimens (Mahony et al., 1997). The development of multiplex PCR tests may prove to be useful in improving our understanding of the epidemiology of these important sexually transmitted diseases in areas where these are endemic. A denaturing gradient gel electrophoresis (DGGE) fingerprinting of 16S rDNA of 32 mycoplasma species has also been developed for rapid identification of Mycoplasma species, and this can be useful when specific PCR is not available (McAuliffe et al., 2003).

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