RMM Tutorial: Nucleic Acid Amplification Technologies

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Scientific Principles

Nucleic acid and gene amplification technologies (also known as genetic or molecular methods) are now recognized as providing superior results when compared with other microbiology methods for particular types of assays. For example, there is a growing trend that more and more pharmaceutical companies are implementing genetic methods for microbial identification and detection applications due to their greater accuracy, speed to result, automation and throughput as compared with classical methods. And the regulatory authorities have also acknowledged the advantages in these types of rapid methods. The FDA Aseptic Processing Guidance recommends the use of rapid genotypic methods for microbial identification, as these methods have been shown to be more accurate and precise than biochemical and phenotypic techniques, and they are especially valuable for investigations into failures (e.g., sterility test; media fill contamination). Both the European and Japanese Pharmacopeias now recommend PCR for Mycoplasma testing. And the Australian Therapeutic Goods Administration (TGA) requires that a sensitive method of microbial identification, such as molecular typing techniques utilising RNA/DNA homology, is utilized when the identification of an isolate is used to invalidate a sterility test.

Nucleic acid and gene amplification technologies utilize a number of scientific principles, including different types of polymerase chain reaction (PCR), transcription-mediated amplification, 16S rRNA typing (ribotyping), and gene sequencing of specific targets of interest, to name a few. Many of these methods have been designed to rapidly detect the presence of a specific microorganism (e.g., an objectionable or specified “compendial” organism), or generate data that can be used to determine the identification of a recovered microbial isolate, from the Genus level down to the sub-species and/or strain level. Additionally, for systems that utilise amplification platforms, the number of amplification cycles to reach a certain threshold level can now be used to estimate the number of organisms in the original sample. Although there is not enough space to discuss all of the available technologies on this page, an overview of the most commonly used nucleic acid amplification rapid method platforms is provided.

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 immobilized on a nylon membrane (this is commonly referred to as an automated Southern Blot technique).

The double-stranded DNA is then denatured to single-stranded DNA, and the membrane is subsequently hybridized 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 recognized, 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 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 utilize PCR to detect the presence of 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, mold and Mycoplasma.

Reverse Transcriptase 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 synthesizes the cDNA. Next, RNAse H removes the remaining single-strand RNA, and a second primer anneals to the cDNA. DNA polymerase will synthesize 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, mold, 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 dideoxyribonucleotides are used, where the latter nucleotides lack a 3'-hydroxyl (-OH) group on their deoxyribose sugar. When a dideoxyribonucleotide is incorporated during sequencing, elongation of the resulting DNA chain is terminated. This provides DNA fragments of varying lengths.

Because each dideoxyribonucleotide is labeled with a different fluorescent dye, each length of DNA fragment ends with a specific dye. A genetic analyzer then separates all of the fragments (i.e., from smallest to largest) using capillary electrophoresis, and a laser detects the nucleotide fluorescence color from the labeled 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

There exist a wide range of additional systems 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 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. MALDI-TOF mass spectrometry is then performed, and the DNA or RNA fragments reach the detector according to their nucleotide composition and length. The resulting mass spectra can be used for the detection of a specific microorganism or for microbial identification using a mass spectral database.

In the second system, a set of 12 broad-range bacterial PCR primers are used to generate PCR products which are then transferred to an electrospray ionization-TOF mass spectrometer. The resulting spectral data are compared with an internal database for bacterial or viral identification.

Summary

The number of available nucleic acid and gene amplification-based rapid microbiological methods has increased over the last few years, and for good reason. They provide an accurate and reliable means for detecting specific microorganisms of interest, especially in pharmaceutical dosage forms that are required to be free of objectionable or specified pharmacopeial organisms. Furthermore, genetic-based rapid methods are now the preferred choice for microbial identification when a contamination event has occurred, due to their enhanced accuracy and reproducibility.

Finally, there are two excellent references on nucleic acid amplification RMMs by Drs. Jimenez and Denoya. Both of their publications are listed on our References Page.