DECONTAMINATION & STERILISATION
biology established after the discovery of DNA, and how it can be replicated and controlled using enzymes. This process of replication is called PCR, or Polymerase Chain Reaction.
The principle of PCR is as follows: The bacteria in a sample are broken open to release their DNA from inside the cell. Short pieces of DNA (primers) are used to ‘find’ the region to be amplified, and new copies of DNA are created using a type of DNA-building enzyme (polymerases). The process is carried out by varying the temperature over a thermal cycle, which takes just minutes. As this thermal cycle, is continuously repeated, the DNA will replicate exponentially over the course of a few hours. This large amount of DNA produced can be easily detected by either fluorescence or separating on a gel through its electrical charge (electrophoresis)
Advantages: PCR can be extremely rapid and very specific. In just a few hours the presence or absence of a single species can be confirmed, along with a quantitative measure of the amount of original DNA (in Genomic Units as opposed to Colony Forming Units).
Disadvantages: The technique must be carried out in the laboratory environment, as DNA contamination is a risk. PCR will also detect dead bacteria that don’t necessarily present any infection control risk, although techniques have been developed which go some way to address this issue. Because of the various techniques employed by each laboratory, it is very difficult to compare results, and there is no ‘standard’ method to judge compliance. Sensitivity is a major problem, although PCR can theoretically detect single bacteria. Because of the small volumes used in the method, bacteria may need to be in the tens or hundreds in a water sample to be detected reliably. In the healthcare setting the approach needs to be flexible, and the impact on the decontamination process should be considered, as shutdown due to failed results and/or major refurbishment could have “serious consequences for patient care” (HTM 01-06 Part E, 2016). Unless cost is prohibitive, these methods can be run side by side with the approved methods to give a complete picture and provide additional assurance. The user needs to make a decision based on the urgency of the results, and take a risk- based approach before using PCR to replace well-established plate-based microbiology to detect bacterial pathogens. PCR is not the only rapid molecular biology method, but it is the most common; other techniques involving enzymes and antibodies are emerging, but none that have the flexibility of the PCR technique. The assay can be adapted to
134 Health Estate Journal October 2019 Bacterial cell
Denaturation (94˚C)
Lysis of cell to free double stranded DNA
New copies of the DNA created
Annealing of primers (54˚C)
Extension (72˚C)
The PCR process: The denaturation, annealing, and extension, are repeated multiple times to create large amounts of the specific DNA section of interest.
not only quantify the amount of DNA, but specific species and groups of organisms can be isolated based on their unique genetic material.
Protein
Testing for protein is crucial on reusable medical devices, as it provides a valuable indicator of cleaning efficacy. Proteins will be introduced to the external and internal surfaces during patient procedures, and will mainly be comprised of blood components – albumins, globulins and fibrinogen, and components from mucosa – mucins, glycoproteins, and various enzymes. All proteins are composed of amino acids; there are 21 types of amino acid, and detection of these (and the bonds between them) forms the basis of most instant protein tests. There are two categories of colour-change type tests – those which use copper ions, and those which use dyes to detect proteins.
Copper-based protein tests The reduction of copper to form a coloured complex forms the basis of the biuret and Lowry methods. These require a peptide rather than free amino acids, as they rely on the structure of the amino acids when in the peptide chain. A modified version of the biuret reaction is the BCA (bicinchoninic acid) test, which responds more uniformly to different proteins at higher levels of sensitivity. The BCA method is one of the most common laboratory-based tests for quantifying protein.
Other protein tests
There is an abundance of different dye- based assays, but the most common visually assessed test is the Coomassie blue/Bradford method, which is simple to carry out in a test-kit form at room temperature without incubation. This dye
was originally developed for textiles, but now forms the basis of most protein test kits. This has replaced the Ninhydrin test, which was found to be not sensitive enough to demonstrate the <5 μg sensitivity required by the latest guidance. In the quest for more instant and enumerable methods some alternative tests have been employed. The Coomassie blue and ninhydrin test kits are often limited, as they are not quantitative (some are semi-quantitative by visual colour comparison). This has led to the popularity of ATP-based methods as a measure of cleaning efficacy.1
Although
ATP does provide some measure of contamination, as it will indicate current and past contamination from living organisms, ATP (adenosine triphosphate) is a universal energy molecule, and is derived from a DNA nucleotide and in no way can be considered a protein. It is important that this is not used in place of a test where a protein is specifically required to be measured, as with surgical instruments.
Some methods must be read using UV excitation and detection of fluorescence. OPA (ortho-phthalaldehyde)-based reagents have been used to detect proteins, peptides, and amino acids, for over 30 years in the laboratory.2
this has been applied to surgical instruments for a more direct measure. When the reagent is applied as a spray, it is possible to detect the protein in situ without needing to swab the instrument; this method of imaging is quantifiable, based on the intensity of fluorescence.
Choosing a protein test When purchasing a protein test kit, in addition to cost and ease of use, it is important for the user to consider: n Sensitivity, ie, the lowest detection
Recently,
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