MICROBIOLOGY SERIES


commonly used genetic-based rapid method platforms available today.


Ribotyping To maintain correct RNA structure and ribosome function in bacteria, the 16S sequence of rRNA is highly conserved at the Genus and species level. This is one reason why Bergey’s Manual of Systemic Bacteriology now uses 16S rDNA sequences as the standard for taxonomic classification of bacteria. However, the non-conserved fragments within the rRNA operon (the spacer and flanking regions of the 16S sequence) can be used to differentiate strains within a particular species. One RMM technology makes use of the DNA sequences that encode for the rRNA operon for microbial identification and strain differentiation. In this fully-automated system, DNA is extracted from a pure culture of bacteria (e.g., using heat inactivation and lysis agents). The extracted DNA is then cut into smaller fragments using restriction enzymes, such as EcoRI or PvuII. The fragments are then separated according to size by gel electrophoresis and immobilised on a nylon membrane (this is commonly referred to as an automated Southern Blot technique). The double-stranded DNA is denatured to single-stranded DNA, and the membrane is subsequently hybridised with a DNA probe (derived from an E. coli rRNA operon). Finally, an antibody-enzyme conjugate is bound to the probe and a chemiluminescent agent is added. Light emitted by the fragments is captured, and the image or banding pattern is compared with patterns stored in the system database. If the pattern is recognised, a bacterial identification is provided. The pattern can also be used to determine if the same strain has been previously observed. This may be helpful when investigating the source of an environmental isolate or a failed microbiology event, such as a positive sterility test or contaminated media fill.


Polymerase Chain Reaction (PCR) A number of RMM detection and identification systems employ different types of PCR as their underlying core technology. In a classical PCR reaction, DNA is extracted and heated to separate the double strands. DNA primers (short, synthetic sequences) are added, which bind to unique target sequences on the template DNA, if they are present. A heat-stable DNA polymerase, such as Taq DNA polymerase, and nucleotide bases (i.e., A, T, G, C) are then


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European Pharmaceutical Review Volume 16 | Issue 5 | 2011


added. The primer is elongated, producing two new complete copies of the template DNA strands. This process is repeated, resulting in millions of copies of the target DNA in a short period of time. Real-time, quantitative PCR measures the DNA amplification reaction as it occurs, while providing an understanding of the amount of target DNA that was in the original sample. In this instance, we can also correlate the number of amplification cycles with an estimation of the number of microorganisms in the sample, in addition to obtaining information about the presence of specific microbial species. There are a number of commercially available rapid method systems available today that utilise PCR to detect the presence of


acid and gene amplification-based rapid microbiological methods has exploded over the last few years, and for good reason”


“The number of available nucleic


certain types of microorganisms as well as to estimate viable cell counts. A variety of primers and detection probes also make multiplexing, or the ability to detect more than one DNA target at the same time, a reality. Many of the systems are now semi- or fully-automated, and the types of organisms that can be detected are broad-based, and include bacteria, yeast, mould and Mycoplasma.


RT-PCR A modification of the classical PCR reaction utilises RNA as a starting template for the PCR reaction, instead of DNA. Here, the enzyme Reverse Transcriptase (RT) will convert extracted RNA into a complimentary strand of DNA (cDNA). For example, a primer first anneals to the target RNA sequence, if present. Then, RT synthesises the cDNA. Next, RNAse H removes the remaining single-strand RNA, and a second primer anneals to the cDNA. DNA polymerase will synthesise the second cDNA strand, resulting in double- stranded cDNA, which is then used in the classical PCR reaction. The reason why RT-PCR is so powerful is that RNA is a better marker of cellular viability than DNA, because RNA is not as stable outside of the cell as DNA is. Additionally, there is less risk of detecting (and amplifying) DNA from non-viable cells or residual DNA from the sample and/or work environment. RT-PCR is now being used for the detection of specific types of microorganisms and the estimation of viable cell count.


Gene sequencing Gene sequencing has been around for some time, but with the availability of automated instrumentation, the pharmaceutical industry has recently taken a more interested position in using this platform for the accurate identification of bacteria, yeast, mould, Mycoplasma and other organisms. The basic premise behind the method is to sequence the first 500 base pairs of the 16S rRNA gene for bacteria or the D2 region of large-subunit rRNA gene for fungi. DNA that has been extracted from a pure


culture of microbial cells is first amplified using classical PCR. Primers direct the sequencing of the forward or reverse reaction for each of the PCR-amplified DNA strands. A mixture of standard nucleotides and dideoxyribo - nucleotides are used, where the latter nucleo - tides lack a 3'-hydroxyl (-OH) group on their deoxyribose sugar. When a dideoxyribo - nucleotide is incorporated during sequencing, elongation of the resulting DNA chain is terminated. This provides for DNA fragments of varying lengths. Because each dideoxyribo - nucleotide is labelled with a different fluorescent dye, each length of DNA fragment ends with a specific dye. A genetic analyser then separates all of the fragments (i.e., from smallest to largest) using capillary electrophoresis, and a laser detects the nucleotide fluorescence colour from the labelled dideoxyribonucleotide at the end of each fragment. Therefore, the actual DNA sequence is based on both fragment size and fluorescence. Finally, the system’s software compares the resulting sequence with the rDNA database and if a sequence match is found, the Genus and species identification is provided.


Additional nucleic acid RMMs A wide range of additional systems exist that are commercially available. Two new technologies combine PCR and mass spectrometry for microbial identification. Since the exact mass of each of the bases which comprise DNA or RNA are known with great accuracy, a high precision measurement (of PCR target sequences) obtained via mass spectrometry has been used to derive a base composition. In the first system, primers composed of a variety of sequences, including housekeeping genes (multi-locus sequence typing; MLST) and 16S rDNA, are used. Genetic material extracted from a sample is used to generate PCR target sequences, and these sequences are transferred to a silicon chip.


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