High-throughput qPCR

Ever-faster, ‘high-throughput qPCR’ methods are becoming available for the simultaneous detection and identification of large quantities of genes. This study looks at how a number of promising methods compare with the qPCR methods already being used by KWR, with a focus on antibiotic-resistance genes in water. We are looking at applicability, quality, potential and costs.

High-throughput versus conventional qPCR

Developments in molecular methods for the detection and typing of microorganisms are moving extremely quickly. A few years ago, a separate (q)PCR method was used for each bacterium; at present, people are using high-throughput qPCR, which allows many bacterial species, types and genes to be analysed at the same time. This development, which allows the activity of very large numbers of genes to be screened simultaneously, is primarily driven by the medical and pharmaceutical sciences. The approach is also used in clinical labs to screen patient material for large numbers of bacterial or antibiotic resistance genes.

Suppliers of diagnostic tests are currently developing test systems and/or platforms that can be used, for example, to screen dozens or hundreds of resistance genes or pathogens in a single sample. In the drinking water sector, where many individual qPCR methods have also been developed in recent years, we are now seeing the need to boost screening capacity with a view to resistance genes. If high-throughput qPCR turns out to work well on concentrates of water samples, that means we will have a method within reach that can be used for the simultaneous detection of a large array of, for example, pathogens, indicator organisms and source tracking markers.

There is an urgent need in the water sector for the simultaneous screening of multiple resistance genes. We are therefore focusing for this purpose on acquiring and testing the high-throughput qPCR method in this pioneering project. In addition, suppliers are already marketing test platforms for this method that are suited to our current equipment and we have reference methods in-house against which we can compare high-throughput qPCR. More specifically, both prefabricated platforms – with resistance genes determined by the supplier for testing – and open platforms – with minimal selection for the testing of resistance and other genes – will be studied in terms of applicability, quality, potential and costs.

Selecting and testing a range of methods

First of all, we determine on the basis of the literature which clinically relevant antibiotic resistance genes and mobile genetic elements are found in water and should, ideally, therefore be included in the platform. In addition, integrons and controls such as the 16S gene (generic) are also included in this overview.

Next, on the basis of scientific literature and information from suppliers, we will look at which platforms are relevant and available for the detection of a series of resistance genes with high-throughput qPCR. Initially, we will concentrate on platforms that are suitable for the BioRAD qPCR CFX96 thermocyclers at KWR’s disposal. During this review, we will focus on the number of relevant resistance genes and mobile elements, the volume to be studied, quality control, the supplier, costs, references for use in clinical and/or water/environment/food research.

In the next phase, we will conduct tests with one or more platforms for high-throughput qPCR systems selected on the basis described here. We will use bacteria that carry known, specific resistance genes. If necessary, cultivation methods for these resistant bacteria will be procured. High-throughput qPCR will be used to study whether, and at what level of sensitivity, the DNA of the bacteria in question can be detected. In addition, DNA will be extracted from sewage-water samples to determine which resistance and mobile genes are present using high-throughput qPCR. Various dilutions of bacterial cultures with known resistance genes will then be added to sewage water. By examining this ‘spiked’ sewage water with high-throughput qPCR, it will be possible to determine whether the known resistance genes can be detected, and down to which dilution. A detailed protocol will be drawn up for this procedure. If possible and opportune, biofilm samples will also be examined in the same way as the sewage-water samples.

Evaluation of application potential

The aim of this project is to evaluate the potential of high-throughput qPCR for use in the sector study (BTO) and at the water companies and laboratories. It therefore represents the next step in the development of DNA-based detection methods. Given the potential of high-throughput qPCR to analyse dozens or even hundreds of DNA targets simultaneously, a new generation of monitoring methods of water quality is now within reach that can be used to determine a whole range of relevant microbiological parameters in a single analysis.