Key breakthroughs in molecular POC testing

Molecular point-of-care (POC) testing is rapidly evolving, allowing for faster and more accessible diagnostics. Learn more about the emerging technologies used and the sample preparation techniques that are key to ensuring their success.

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Moving from lab-based to POC testing

Molecular POC. When you hear it, you may imagine a sensitive and specific test conducted at a patient’s side. You can see it accelerating clinical decision making and improving the patient's outcome. But among the positive thoughts, phrases like “complex” or “time-consuming” or “technical” might also pop into your mind. Indeed, these downsides were previously roadblocks to moving molecular diagnostics from lab-based assays to bed-side tests.

However, thanks to recent advances, molecular testing to detect nucleic acid disease biomarkers is now being adapted for use outside of laboratories. These advances include the innovative use of amplification techniques and microfluidic technology and improvements in sample and reagent preparation such as liquid biopsy and lyophilization.

Emerging molecular POC technologies

Adapting molecular diagnostics techniques for use in POC testing has been no easy feat, with the need to increase the speed, portability, and simplicity of lab-based techniques; all while retaining high sensitivity and specificity.

A key breakthrough in POC testing has been the use of microfluidic technologies to create “lab-on-a-chip” POC assays. These assays complete all sample, analysis, and detection reactions within microfluidic channels. As a result, lab-on-a-chip assays are small, portable, and require very little sample and reagent. They drastically reduce costs and turnaround times and can enable the detection of multiple analytes.

Several types of diagnostic techniques have successfully been used in molecular POC tests, including polymerase chain reaction (PCR), isothermal amplification techniques, and CRISPR.

Faster, more portable PCR testing

PCR, particularly reverse transcriptase-PCR (RT-PCR) and real-time PCR (qPCR) are the gold standard techniques in molecular diagnostics. These techniques can be extremely sensitive, with the ability to amplify and produce a detection signal from a single DNA or RNA molecule.

Several RT-PCR systems that use microfluidic technology including the Cepheid GeneXpert have been granted the clinical laboratory improvements amendments (CLIA) waiver by the United States Food and Drug administration (FDA), allowing their use in POC settings. These systems typically use a single cassette that contains separate chambers for sample preparation, amplification, and detection.

The small reaction volumes used in these microfluidic systems vastly reduce the time required for thermocycling, providing results within 30 minutes. So far, the GeneXpert system has been used in many tests, including for the multiplex detection of COVID-19, influenza A and B, and the multiplex detection of several human papillomavirus (HPV) oncotypes (1–3).

As microfluidic technology and other test components advance, we should expect to see a further increase in the speed of PCR-based POC tests (Fig 1).

Fig 1. PCR system designed with a microfluidic chip

Rapid and simple POC testing using isothermal amplification

Isothermal amplification methods are arguably better suited to POC testing than PCR, with low-cost, single-tube assays that don’t require thermocycling steps. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are the two most commonly used isothermal amplification techniques in molecular POC tests.

LAMP-based devices

In recent years, several LAMP systems with a reverse transcription step (RT-LAMP) have been increasingly used in POC settings. These systems are table-top platforms with kits available for detecting various infectious diseases. A user simply mixes a patient sample with kit reagents as directed, places tubes into the machine for amplification, and receives results in less than an hour. The tests can even be done without extracting nucleic acids from the sample. Diseases that can be diagnosed include malaria, strep A and strep B, HPV, whooping cough, and sexually transmitted infections within just 30 minutes (4, 5).

The development of LAMP-on-a-chip systems is further improving the speed and accessibility of LAMP-based testing (Fig 2). These devices can detect multiple HPV types in just 15 minutes and have even been used to detect COVID-19 within just 3 minutes, being the fastest COVID-19 test reported as of 2023 (6, 7).

Fig 2. The key steps and components for the Integration of LAMP with microfluidics

Lateral flow strips integrating RPA

RPA is another rapidly advancing molecular diagnostic technique used in POC testing (Fig 3). Its main advantage is the low isothermal amplification temperature of 37°C to 42°C, with amplification even possible at room temperature.

Another key advantage of RPA is that the labeled amplification products can be easily detected using lateral-flow strips. So you get the benefits of lateral-flow tests, including their low-cost, simple components, and rapid development along with RPA’s high accuracy and fast turnaround times.

RPA lateral-flow tests have been used to detect several infectious diseases such as COVID-19, Salmonella, the tropical disease Schistosoma mansoni, and the multiplex detection of Dengue virus serotypes (8–10).

Fig 3. (A) Schematic Diagram: Illustrating the fusion of RPA with LFD, featuring FAM (carboxyfluorescein) marker. (B) Diagrammatic Sketch: Outlining the structure of LFD, with test and control lines. (C) Results: Depicting positive and negative outcomes.

Combining isothermal amplification with CRISPR

Despite the many positives of isothermal amplification-based POC tests, a major downside is their risk of nonspecific amplification, which could potentially lead to false positive results. To overcome this, scientists are developing POC tests that integrate isothermal amplification with the highly accurate gene editing technology, CRISPR.

In these tests, DNA is first amplified using isothermal amplification and then detected using CRISPR/Cas systems. And it all happens in a single tube. A range of different CRISPR-isothermal POC tests have been developed including those to detect COVID-19, malaria, and African swine fever. These tests have shown high performance, with some even boasting 100% specificity and sensitivity (11–13).

The power of sample and reagent preparation

The transition of molecular diagnostics from lab to POC has been aided by advancements in sample and reagent preparation steps. Various techniques have been developed to streamline and enhance cell and nucleic acid isolation, purification, and reagent stability. These techniques include liquid-based cytology, liquid biopsy, and the use of true pore membranes such as track-etched membranes, aluminum oxide membranes, and lyophilized reagents.

Liquid-based cytology and liquid biopsy for cancer diagnoses

Liquid-based cytology has emerged as an innovative sample preparation technique for cancer diagnostics. This process involves the suspension and fixation of cells in a liquid medium, along with the removal of any cellular debris and contaminants. Monolayer slide preparation technology is used to prepare cells for microscopic analysis, providing an effective first-line cancer diagnostic that is commonly used prior to molecular testing.

The cell samples prepared using liquid-based cytology can also be used in downstream molecular POC tests (14). However, the fixatives used, such as formalin and formaldehyde, must first be removed to prevent their interference with diagnostic tests.

Another sample preparation technique that is revolutionizing cancer diagnostics is liquid biopsy. This process entails the isolation of tumor-derived materials, including circulating tumor cells, circulating tumor DNA, and extracellular vesicles from blood or other bodily fluids. Liquid biopsy is a convenient, quick, and noninvasive alternative to conventional tissue biopsies. It requires very little sample, allowing for the development of POC cancer diagnostics.

Read more on the role of liquid biopsy in cancer diagnosis and treatment

Track-etched membranes

Membrane-based POC devices are increasingly used in molecular diagnostics for their speed, robustness, affordability, ease of use, and scalability. However, their performance relies heavily on the choice of materials used. The membranes for sample preparation are one of the most important components, responsible for separating and purifying the target analyte.

Track-etched membranes (TEMs) are one of the best choices for sample preparation in molecular POC tests. Manufactured from high-quality polycarbonate (PC) or polyethylene terephthalate (PET) film, TEMs have high flow rates, excellent chemical and thermal resistance, and well-defined pore sizes.

Their controlled pore diameter allows for the accurate separation of the target analyte from any contaminants or inhibitors. When integrated into POC tests, TEM membranes offer fast and effective sample filtration, with very little loss of the analyte.

Learn more about the properties and applications of track-etched membranes

Lyophilized reagents

An obstacle faced in the adaptation of molecular diagnostics from the lab to POC has been the stabilization of the reagents used. Many are unstable at room temperature, requiring storage in -80°C freezers. This temperature requirement limits the transport and storage options for molecular tests in POC settings.

The issue of reagent stability can be solved with lyophilization, also known as freeze-drying. This process reduces the water content of reagents to make them less reactive and more stable at room temperature. POC test kits using lyophilized reagents are easier to store and transport. They also simplify molecular POC assays because single-dose reagents to be combined within a single tube in a reagent pellet.

Read more on the benefits of lyophilization in assay development

The bright future of molecular POC testing

The evolution of molecular diagnostics techniques from lab-based settings to the patient’s side marks a significant milestone in healthcare. The integration of these techniques into POC devices has enabled the development of portable, ultrafast diagnostic tests across a broad range of applications.

Looking to the future, ongoing advancements in POC test development are predicted to further increase the speed, performance, and applications of diagnostic tests, from infectious disease to oncology and beyond.

As POC testing evolves, Cytiva can help you navigate the translation of complex diagnostic concepts into reliable POC assays. Our broad range of custom diagnostics services can help you accelerate the development of your molecular diagnostic assays, from ideation right through to commercialization.

Visit our custom diagnostic services page to find out more, or request to speak with a specialist