Multiplexing in point-of-care diagnostics

An ideal POC test meets the ASSURED criteria: Affordable, sensitive with minimal false negatives, specific with minimal false positives, easy to use with minimal steps to perform, robust, device-free - no complex equipment required and available to the end user. It is often necessary to differentiate between several pathogens, in which case it is a great advantage to be able to detect several pathogens simultaneously from one sample (multiplexing). This saves time and resources and allows for a more comprehensive, faster diagnosis directly at the point of care.

Rapid diagnosis directly on site: POC tests

A wide range of technologies are available for the laboratory and device-based diagnostic detection of pathogens. Most commercially available detection methods are based on the classical methods of enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR). Despite their high specificity and sensitivity, their use as POC tests is hampered by the need for high-tech equipment, specialized laboratories, trained and experienced personnel, and high costs. In addition, the specificity and sensitivity of all immunological methods such as ELISA depend heavily on the quality of the antibodies used.

In contrast to laboratory-based pathogen detection, POC tests offer the advantage of being able to make diagnoses particularly quickly, which is crucial in clinical situations. However, this speed must not come at the expense of reliability. POC tests are sometimes less accurate than their laboratory counterparts. It is important that the sensitivity of the tests used is significantly better than required for diagnosis. When using POC tests, it should be considered whether their use is justified despite possible inaccuracies.

The test we have developed based on the “Loop-mediated Isothermal Amplification” (LAMP) detects specific nucleic acid sequences of the pathogens with high sensitivity. For the detection of SARS-CoV-2, for example, we showed a sensitivity 10 to 100 times better than required for diagnosis.

Another advantage of POC tests is their ease of use. The tests are designed to be performed by less experienced personnel, which facilitates their use in resource-limited settings. Our POC test does not require complex equipment or time-consuming pathogen purification, but trained personnel is advantageous to ensure the reliability of the results.

Simultaneous detection: multiplex tests

Multiplex technology allows for the simultaneous detection of multiple pathogen sequences in a single sample, increasing efficiency. By analyzing multiple target sequences simultaneously, time and sample material are used more efficiently, reducing the cost per test.

Performing multiplex testing can be complex and requires careful planning. LAMP technology uses six different primers per detection, requiring a high level of expertise in primer set design to avoid mutual interference. Differences in the efficacy of LAMP reactions can lead to dominance of the most sensitive detection and increase the risk of cross reactivity.We use lateral flow dipsticks (LFD) to visualize the results. They are easy to handle and enable the specific detection of positive results, which is not possible with most POC-suitable LAMP detection systems. With LFDs, this is achieved by the specific binding of the labeled fragments to the test strip.

The maximum number of pathogens in a multiplex assay is not only limited by the complexity of the reaction set-up, but also by the number of test lines on the available LFDs. During the development of our assay for the detection of potato viruses in the “TRV2Go” project, only LFDs with a maximum of two addressable test lines were available, which allowed a maximum of duplex reactions. Therefore, in the “2detect” project, we commissioned the development of a test strip with five test lines to enable the simultaneous detection of up to four different labeled LAMP amplification products and a target line for internal LFD function control. In our test, we omitted one of the test lines in favor of an additional control that uses amplification of a housekeeping gene to ensure that sampling, setup of the LAMP reaction and analysis of the results by the LFD were performed correctly to detect false positives and false negatives. We have successfully used these test strips for the detection of labeled amplification products of various human pathogenic respiratory viruses (RSV; Influenza A; Influenza B; SARS-CoV-2). In future projects, we are also planning to use the test strips for the differentiated detection of other pathogens, for example from livestock or crops.

Conclusion

The new LAMP test procedure meets the ASSURED criteria and offers significant advantages in the field of multiplex diagnostics. The simultaneous detection of multiple pathogens from a single sample enables an efficient and comprehensive analysis saving time and resources. The high sensitivity of the LAMP test, especially in the detection of respiratory viruses, ensures the reliable identification of even rare pathogens Its ease of use and avoidance of complex equipment make it particularly attractive for use in resource-limited areas and emergency situations. The newly developed test strip with five test lines enables the simultaneous detection of up to four pathogens and contributes to fast, precise and cost-efficient diagnostics.
The advances in multiplexing position our LAMP as a breakthrough solution in pathogen diagnostics that meets the needs for affordability, sensitivity, specificity, ease-of-use, robustness and availability