06/​15/​2021

Milenia HybriDetect – Nucleic Acid Amplification and Lateral Flow

I have been working on universal lateral flow devices (especially nucleic acid ampli­fi­cation and lateral flow) for more than eight years. Since then, I try to get an overview of the variety of appli­ca­tions, test them and make them more acces­sible for simple and rapid lateral flow readout. After all these years, experi­ences and, of course, failures, the enthu­siasm for this appar­ently simple platform is still unbroken. There are no limits for individual creativity, because there is so much charme in the simplicity of the lateral flow technology. There are countless ways to think up, modify and optimize the test strips and the associated methods. For me personally, Nucleic Acid Lateral Flow Immunoassays (NALFIAs) are the ultimate playground for creativity. And finally, innov­ative test systems with excellent precision and sensi­tivity can be created.

The following article is intended to describe an important part of the “lateral flow-​​world”: the combi­nation of nucleic acid ampli­fi­cation and lateral flow.

More than just a Platform for Nucleic Acid Ampli­fi­cation and Lateral Flow

Milenia® HybriDetect is a universal Lateral Flow Devel­opment Platform that is cited in more than 145 publi­ca­tions. The overwhelming majority of these peer reviewed papers decribe quick and easy identi­fi­cation of DNA-​​amplification products. This method­ological combi­nation of DNA-​​/​ RNA-​​amplification and lateral flow allows the decou­pling of complex methods from specialized labora­tories and thus the devel­opment of field deployable tests. Although the following sections refer specif­i­cally to nucleic acid ampli­fi­cation techniques, the Milenia HybriDetect can be more than just an amplicon detection format.

Learn more about Lateral Flow Basics, Milenia HybriDetect Compo­sition and Detection Principles

 

How to handle the Milenia® HybriDetect

The general processing of the Lateral Flow Analysis remains almost the same regardless of which appli­cation is combined with this procedure. The sample can be applied directly to the strip or into the assay buffer (Fig.1, 1 – 3). The test strips are positioned verti­cally in a volume between 50 and 100 µL, which initiates the lateral flow (Fig.1, 4). Recog­nizable signals can appear after a few seconds. The results are usually inter­preted after 2 – 10 minutes (Fig.1, 5).

Figure 1. General processing of the Lateral Flow Analysis using the Milenia HybriDetect® Lateral Flow Assay Kits

Polymerase Chain Reaction and Lateral Flow

The polymerase chain reaction is the frist described, best under­stood and most important DNA-​​amplification method worldwide. Ultimately, all types of DNA-​​amplification techniques are based on this technology. Never­theless, PCR is a relatively under­rep­re­sented technique for point-​​of-​​care appli­ca­tions. PCR is often described as laborious, expensive and time intensive. Furthermore, PCR needs special equipment and highly trained personnel. These arguments are admit­tedly one-​​sided and neglect the important advan­tages of this method. The simple assay design, the method­ological versa­tility, the multiplex compat­i­bility, the devel­opment in reagents and the large number of already estab­lished PCR-​​variations make this method an absolutely under­rated technique in the field of point-​​of-​​care testing.

Polymerase Chain Reaction-based DNA-Amplification and Lateral Flow
Figure 2. Combi­nation of Polymerase Chain Reaction and Lateral Flow

Recom­binase Polymerase Ampli­fi­cation and Lateral Flow

The Recom­binase Polymerase Ampli­fi­cation (RPA) uses beside a polymerase with strong displacement activity special compo­nents (recom­binase, ssDNA-​​binding protein, loading factor, crowding agent) to allow specific, isothermal ampli­fi­cation at lower temper­a­tures between 37 and 42°C. The RPA is an extremely fast DNA-​​amplification technique, that is very robust against several inhibitors. Taken together, the RPA is one the most popular methods for fast, sensitive, specific and field-​​deployable DNA-​​amplification. The combi­nation of the recom­binase polymerase ampli­fi­cation and lateral flow based amplicon detection allows the devel­opment of almost equipment free rapid tests that are partic­u­larly useful in appli­ca­tions under low-​​resource-​​settings.

Recombinase Polymerase Amplification and Lateral Flow
Figure 3. Combi­nation of Recom­binase Polymerase Ampli­fi­cation and Lateral Flow

Loop mediated isothermal Ampli­fi­cation and Lateral Flow

The loop mediated isothermal ampli­fi­cation (LAMP) is one the most frequently used isothermal DNA-​​amplification methods. The use of multiple primers in combi­nation with a DNA polymerase with strong strand displacement activity and constant temper­a­tures of 60 – 65°C allow rapid and sensitive DNA-​​detection. One charac­ter­istic of the LAMP is the method­ological diversity allowing extremly sensitive assays or advanced speci­ficity (e.g. SNP-​​detection). The combi­nation of multiplex LAMP with a lateral flow-​​based amplicon detection is one the most promising tools in POC-​​DNA-​​diagnostics so far. Learn more about LAMP and Lateral Flow

loop mediated isothermal amplification and lateral flow
Figure 4. Combi­nation of Loop Mediated Isothermal Ampli­fi­cation and Lateral Flow

CRISPR/​Cas-​​dependent Nuclease Assays and Lateral Flow

In most cases these methods combine isothermal ampli­fi­cation methods, like LAMP, RPA or NASBA with a specific CRISPR/​Cas based nuclease assay. Two consec­utive enhancer reactions result in ultra­sen­sitive diagnostic appli­ca­tions that are partic­u­larly inter­esting for detection of viral pathogens. The specific breakdown of dual labeled reporter is detectable with the Milenia® HybriDetect. The combi­nation of sensi­tivity and speci­ficity of CRISPR /​ Cas-​​dependent assays makes this compar­a­tively new approach to one the most exiting topics in the field of molecular diagnostics.

  • CRIPSR/​Cas 12 – based detection, e.g. DETECTR
  • CRISPR/​Cas 13 – based detection, e.g. SHERLOCK, INSPECTR
  • CRISPR/​Cas 14 – based detection (future appli­cation)
  • CRISPR/​Cas 3 – based detection (principle shown)
CRISPR dependent nucleic acid detection via lateral flow using collateral nuclease activity
Figure 5. CRISPR/​Cas-​​based Reporter Degra­dation combined with Lateral Flow

CRISPR/​Cas dependent „Amplicon Binding Assays“ and Lateral Flow

In most cases these methods combine DNA ampli­fi­cation with a CRISPR/​Cas-​​based amplicon  „binding assay“. The benefit of this method­ological combi­nation is the impressive speci­ficity of the CRISPR/​Cas-​​mediated amplicon „recog­nition“. A striking example is a preprint, presented in february by Kumar and colleages. Cas9 from Fransi­cella novidica is used for identi­fi­cation of SARS-​​CoV-​​2 mutant strains due to specific amplicon recog­nition.

CRISPR dependent nucleic acid detection via lateral flow using specific amplicon binding
Figure 6. CRISPR/​Cas based Amplicon Binding Assay combined with Lateral Flow

More Promising Tools for Nucleic Acid Ampli­fi­cation and Lateral Flow

Today, there are many different varia­tions of isothermal techniques that combine nucleic acid ampli­fi­cation and lateral flow. The focus in the liter­ature is very much on LAMP and RPA. But other classic isothermal techniques such as helicase dependent ampli­fi­cation (HDA), rolling circle ampli­fi­cation (RCA), cross priming ampli­fi­cation (CPA) or strand displacement ampli­fi­cation (SDA) are perfectly compatible with a simple lateral flow readout. But also less frequently used methods such as polymerase spiral ampli­fi­cation (PSA) or nucleic acid sequence based ampli­fi­cation (NASBA) hold great potential in the context of nucleic acid based rapid testing.

It gets partic­u­larly inter­esting if innov­ative molecular tools like the CRIPSR-​​Cas „machinery“, additional DNA-​​modifiying enzyms or DNAzymes are combined with existing DNA-​​or RNA-​​amplification methods. A good and compre­hensive knowledge of the toolbox is the basis for the devel­opment of innov­ative assays formats & nucleic acid detection platforms. The following image summa­rizes important parts of this toolbox for nucleic acid lateral flow immunoassays.

Overview most frequently used DNA-/ RNA dependent detection startegies combined with Milenia HybriDetect
Figure 7.  Overview of the most frequently used nucleic acid dependent methods combined with Milenia® HybriDetect

Outlook – New Methods, Alter­native Techniques, Future Trends and Appli­ca­tions

Innov­ative molecular detection techniques such as SHERLOCK, FELUDA or LEOPARD rely on the use of  the CRISPR /​ Cas technology. Regardless of the future impor­tance of these new tools, they clearly show what the challenges of a new gener­ation of molecular diagnostics are. On the one hand, it is about finding ways to take laboratory analysis to a new level. Especially high throughput analyzes that provide much more infor­mation than a simple “positive” or “negative” are certainly the focus of modern molecular analytics. On the other hand, the SARS-​​CoV-​​2 pandemic has clearly shown the impor­tance of a simple, robust and yet sensitive and precise analysis under low resource settings. Preventive “at-​​home-​​testing” in the SARS-​​CoV-​​2 pandemic has shifted the awareness of diagnostics outside of specialized labora­tories. If you take a look at the initial publi­ca­tions of the above-​​mentioned methods, it becomes apparent that point-​​of-​​care analysis plays a partic­u­larly important role.

Because sensitive, molecular diagnostics must also work outside of specialized labora­tories. This is exactly where the compat­i­bility with extremely simple, robust, instrument-​​free downstream analytics such as the lateral flow technology is one of the keys to modern POC diagnostics.

The third and last part of this blog series is dedicated to give more infor­mation about existing fields of appli­cation and thus highlights more trends and challenges of nucleic acid lateral flow immunoassays.