From traditional methods to realtime PCR in microbiology

Prior to development of the recent molecular diagnostic technologies and their applications in microbiology laboratories, traditional diagnostic methods were used for qualification and quantification of the microorganisms. Direct counting of individual cells by microscopy from samples or culture plates was probably the oldest technique used for microbial quantification. DNA binding stains were also used for the same purpose (Jansson and Prosser, 1997). As enumeration has been of interest, many different cell detection techniques have been developed including fluorescent tagged antibodies, rRNA targeted fluorescent oligonucleotides, confocal laser scanning microscopy and flow cytometric methods (Jansson and Prosser, 1997). Among the traditional methods culture and culture-based methods were considered as reliable approaches and mostly named as the 'gold standard' for the identification and quantification of bacteria. Besides being time-consuming, traditional methods have some limitations such as poor sensitivity, slow growing or poorly viable organisms, narrow detection ranges, complex interpretation, high levels of background, and non-specific cross-reactions (Mackay, 2004). Traditional microbial culture methods supply valuable data especially for new, uncharacterized or atypical organisms for further studies and epidemiological assessments (Mackay, 2004).

There has been a sharp increase in molecular biological techniques over the last few decades, which has resulted in developments in many areas of the life sciences, including bacteriology (Millar and Moore, 2004). The field of molecular biology has been revolutionized by amplifying as few as one copy of a gene into billions of copies, after the introduction of PCR. PCR-based methods offer high sensitivity, specificity and simplification of testing by process automation and incorporation of non-isotopic detection (Sintchenko et al., 1999). In the early stages, the use of PCR methodology was limited by the qualitative nature of PCR-based applications. The logarithmic amplification of the target sequence in PCR reveals with no correlation between the initial and final amounts of DNA product. Due to this limitation, conventional PCR is considered a semiquantitative technique.

Recently, real-time PCR technology appeared as a major development among the PCR-based systems. In these assays, fluorescent signals are generated as the PCR takes place and thus real-time monitoring during the amplification is possible. This useful combination of nucleic acid amplification and signal detection has reduced the time required for nucleic acid detection. In addition, as a quantitative method for detection of bacterial species, sensitivity of real-time PCR enables detection of less than 10 cells with sensitive background regulations (Maeda et al., 2003; Ott et al., 2004). Today, real-time PCR is used to detect nucleic acids from food, vectors used in gene therapy protocols, genetically modified organisms, and areas of human and veterinary microbiology and oncology (Mackay, 2004).

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