Multiplex Applications & Lateral Flow

The combi­nation of nucleic acid analysis and lateral flow technology is without a doubt an important tool for current and future point-​​of-​​care and point-​​of-​​need appli­ca­tions. It doesn’t take a lot of imagi­nation to assess the potential of these techniques. In most appli­ca­tions, the focus is on its simplicity, speed and robustness, whereby the perfor­mance of the analysis can be compa­rable to estab­lished laboratory reference methods. These charac­ter­istics qualify such tests for appli­ca­tions “in the field”. Terms such as speci­ficity, robustness and sensi­tivity are mentioned again and again. But an important point undis­putedly belongs to this list of “test-​​features”. We are talking about the gener­ation of multiple infor­mation per analysis, which is called “Multi­plexing”.

– Multi­plexing is the ultimate challenge in the field of modern diagnostics. It’s much more than just the method­ological “icing on the cake” – Techniques that allow strong multi­plexing without signif­icant loss of sensi­tivity & speci­ficity will be “game-​​changers” for a new gener­ation of field-​​applicable diagnostics – 

Multiplex Appli­ca­tions & the Universal Lateral Flow Platform – Milenia HybriDetect 

Milenia HybriDetect is a universal lateral flow devel­opment platform that allows scien­tists around the world simple rapid test devel­opment. The Milenia HybriDetect family includes two test strips, the Milenia HybriDetect and the Milenia HybriDetect 2T, whereby the 2T strip has 2 test lines and thus allows duplex appli­ca­tions. The multiplex options seem limited, especially for universal test strip formats, but there is actually still a lot of potential hidden in multi­plexing in combi­nation with the use of the universal lateral flow devel­opment platform – Milenia HybriDetect. The following figure describes that a certain degree of multi­plexing can be generated at different points within a NALFIA (Nucleic Acild Lateral Flow Immunoassay. These “levels of multi­plexing” must be perfectly coordi­nated with one another. The structure shown here also serves as a basis for discussion for this article, which is intended to provide an overview of existing technologies combining NALFIAs with multi­plexing and at the same time facil­itate the devel­opment of innov­ative test formats.

Muliplex Applications and Lateral Flow - Levels of Multiplexing
Multiplex NALFIAs — Levels of Multi­plexing


Learn more about the Milenia® HybriDetect – General Detection Strategies

Learn more about the Milenia® HybriDetect – Nucleic Acid Ampli­fi­cation & Lateral Flow

Advantage  – Multiplex PCR & Lateral Flow

The PCR (Polymerase Chain Reaction) is the perfect choice, when it comes to multi­plexing. Due to the simple primer design, simple adaptation of temper­ature protocols and the wide variety of optimized enzyme-​​systems, the PCR generally allows rapid devel­opment of well performing multiplex assays. Although PCR is discussed contro­ver­sially in terms of Point-​​of-​​Care usability, this DNA-​​amplification technique has a lot of untapped potential in the context of NALFIAs. A few selected publi­ca­tions indicate the power of this technology as a basis for further multiplex appli­ca­tions & techniques. These selected publi­ca­tions use the universal Lateral Flow Device – Milenia HybriDetect 2T. [1] [5]

Duplex PCR and Lateral FLow


No DNA-​​Extraction? – DirectPCR and the Impor­tance of an Internal Ampli­fi­cation Control

A wide variety of sample matrices can be used in PCR, if a suitable and robust enzyme system has been selected. This is the prereq­uisite for the direct appli­cation of a biological material in a PCR, elimi­nating the demand for a DNA or RNA extraction prior to the PCR reaction. This is an important feature for the devel­opment of point-​​of-​​care compatible rapid tests. Direct analysis from blood is just one example of practical relevance. The direct PCR format can benefit from the multiplex ability by imple­menting an IAC (Internal Ampli­fi­cation Control. This method­ological approach allows a clear differ­en­ti­ation between negative results and matrix-​​dependent ampli­fi­cation inhibition. The Milenia® HybriDetect 2T allows rapid evalu­ation of a direct-​​duplex PCR. [1] [5]

Importance of an internal amplification control using the direct Polymerase Chain Reaction approach
Impor­tance of an Internal Ampli­fi­cation Control for NALFIAs


We at Milenia Biotec trust in this kind of technology. Therefore, we developed our own PCR-​​Lateral Flow-​​Kits including an extremely robust Polymerase that allows direct analysis without DNA-​​Extraction. Moreover, we developed an optimized IAC as perfect “Inhibition-​​Indicator” and of course, we use our own universal Lateral Flow Device, Milenia® HybriDetect 2T, in order to specif­i­cally detect spoiling bacteria in beer and juice.

Benefit from our experience, knowledge and products. Use the Milenia HybriDetect 2T for rapid devel­opment of duplex PCR appli­ca­tions. Ininitial devel­opment of person­alized PCR-​​LFAs is possible within a few days to weeks.“


More Infor­mation about the Milenia GenLine Kits for rapid detection of Beer Spoiling Bacteria

More Infor­mation about the Milenia GenLine Kits for rapid detection of Alicy­clobacillus spp.

Learn more about PCR & Lateral Flow

Multiplex RPA & Lateral Flow

Generally Recom­binase Polymerase Ampli­fi­cation (RPA) is an isothermal DNA-​​amplification technique that allows extremely rapid and robust analysis. RPA is highly compatible with a simple-​​to-​​handle and –interpret Lateral Flow Readout, but it is not known to be the perfect technique for multi­plexing. Never­theless, it is possible to design duplex RPAs in order to generate two amplicons during the ampli­fi­cation reaction. Especially for this type of appli­cation an optimized Lateral Flow Assay Buffer was designed in order to suppress the disruptive character of RPA reagents against LFD-​​antibodies. This makes the Milenia® HybriDetect 2T the perfect tool for analyzing multiplex RPA products. And this is exactly what has been proven in several scien­tific publi­ca­tions, which are listed below. [2] [6]

Two Infor­ma­tions for singleplex Quantifi­cation: Semi-​​quantitative RPA

Authors from Havard Medical School in Boston became creative with the presen­tation of an innov­ative Recom­binase Polymerase Ampli­fi­cation (RPA) Assay design. They saw a Limitation in existing rapid POC assays because many of them rely on rapid DNA ampli­fi­cation methods, which amplify target-​​DNA extremely fast until saturation effects occur. In conse­quence there is no chance to (semi-​​) quantify the test outcome. In most cases it is a quali­tative “yes” or “no” inter­pre­tation. Therefore, a compet­itive type of RPA was designed. Two differ­ently labeled products are amplified at the same time in the same reaction tube. Amplicons are analyzed with a rapid lateral flow readout, using the Milenia® HybriDetect2T. Combined signal inten­sities of the reference test line and the target test line allow semi-​​quantitative analysis. The more target DNA or RNA is intro­duced into the RPA reaction, the stronger the target test line appears on the LFD. Moreover, the intensity of the reference test-​​line decreases, because the “reference-​​amplicon” is less efficiently amplified. In contrast, if the amount of template decreases, test line intensity decreases as well and the intensity of the reference test line increases. This method­ological approach allows a clear distinction between variable template quantities due to the design of a simple but very clever multiplex RPA variation. [7]

Semiquantitative RPA & Lateral Flow


Link to the original publi­cation by Mancuso et al.

Learn more about RPA & Lateral Flow

Multiplex LAMP

An efficiently performing singleplex LAMP-​​Assay needs 4 to 6 modular designed primers adressing 6 to 8 sequence-​​regions with defined properties & distances. This fact illus­trates the complexity of the devel­opment of a well performing multiplex LAMP. But once a suitable primer combi­nation has been found, the Loop mediated isothermal ampli­fi­cation (LAMP) can be an extremely impressive DNA ampli­fi­cation technique that is fast, highly sensitive, even in a multiplex setup. Beside standard duplex or triplex LAMP, there are more possi­bil­ities to achieve multi­plexing, even with a singleplex Lateral Flow Device.

Two Ampli­fi­cation Products and One Signal – the High-​​Surety LAMP

Researchers from the University of Austin (Texas, USA) describe multi­plexing as an important tool for the reduction of false positive and false negative results. The LAMP, an ideal low-​​tech DNA-​​amplification method for rapid, field-​​deployable testing, was multi­plexed in order to detect SARS-​​CoV-​​2, the causative agent of COVID-​​19. Two estab­lished singleplex LAMP Assays were multi­plexed, which worked out well. Moreover, the researchers developed amplicon-​​specific probes that were able to connect the LAMP-​​products of both reactions. Due to the chosen labeling strategy, only “connected” LAMP-​​products were detectable with the Milenia® HybriDetect on one single test line. As a conse­quence, the presence of two different templates is necessary to generate a positive test result. The presence of just one ampli­fiable template allows ampli­fi­cation, but the Lateral Flow Device will give a negative result. The combi­nation of a duplex LAMP-​​Assay and Lateral Flow Readout allowed sensitive detection of the SARS-​​CoV-​​2 with a signif­i­cantly improved speci­ficity. [3]

High Surety LAMP


Link to the original publi­cation by Bhadra et al.

Learn more about LAMP & Lateral Flow

The Addition of Post-​​Amplification Methods for stronger Multi­plexing

Obviously, commer­cially available, universal Lateral Flow Devices are at some point limited when it comes to multi­plexing. Never­theless, there are ways to achieve a higher level of multi­plexing by using additional techniques for amplicon “recog­nition” in combi­nation with the use of multiple LFDs, in order to generate more infor­mation per analysis. This strategic approach has been described in several peer reviewed publi­ca­tions. The following sections describe three general post-​​amplification methods that may be useful for multi­plexing: simple probe-​​Hybridization, CRISPR/​Cas-​​dependent amplicon-”recognition” & nested ampli­fi­cation.

Post Ampli­fi­cation Methods for Muliplex Appli­ca­tions & Lateral Flow



A very simple approach is the devel­opment of an additional hybridization reaction following the nucleic acid ampli­fi­cation reaction. For each hybridization reaction, 1 or 2 specific probes can be used to “recognize” amplicon-​​specific sequences. By estab­lishing multiple hybridization reactions and using the universal test strips of the Milenia HybriDetect Platform, multiple pieces of infor­mation can be generated in one workflow. In internal exper­i­ments, hybridization in combi­nation with various ampli­fi­cation techniques turned out to be a surpris­ingly powerful method­ological tool, despite its simplicity. From genotyping, discrim­i­nation of SNPs to quite complex species differ­en­ti­ation, the simple hybridization of a labeled probe revealed an unexpected diversity. [1] [3] [6]

CRISPR/​Cas dependent amplicon recog­nition

Over the last few years the CRISPR/Cas-”machinery” turned out to be an extremely valuable and versatile tool for modern molecular diagnostics. Detection sensi­tiv­ities can be signif­i­cantly improved, so that ultra-​​sensitive nucleic acid analyses are possible in a point-​​of-​​care or point-​​of-​​need setting. In addition, these molecular tools also allow an impressive level of selec­tivity. As a conse­quence, diagnostic CRISPR/​Cas-​​dependent methods can achieve outstanding speci­ficity. These properties make this technology a perfect post-​​amplification method that, with a little skill and creativity, can answer even complex questions in field-​​applications. A clear distinction has to be made between various CRISPR-​​Cas-​​related detection techniques. The best-​​known genome editing tool, CRISPR-​​Cas9, is used in the lateral flow context for appli­ca­tions in which gRNA-​​Cas9-​​amplicon-​​complexes are detected on the test of LFD. This principle (label incor­po­ration strategy) is very suitable for multi­plexing. In contrast, with Cas proteins such as Cas12 or Cas13 with high turnover (collateral nuclease activity), the status of a so-​​called dual-​​labeled reporter is analyzed. This enables very sensitive analyzes, which are less suitable for multiplex appli­ca­tions (label separation strategy). The following figure differ­en­tiates between these general method­ological approaches.

Learn more about CRISPR & Lateral Flow

Learn more about general CRISPR-​​based diagnostics (Review from leading scien­tists in the field)

An Example from the Liter­ature: CRISPR/​Cas9 – Multiplex NALFIA

A very inter­esting publi­cation from 2021 by Manoj Kumar, Sneha Gulati and colleagues  illus­trates the use of a SARS-​​CoV-​​2-​​specific ampli­fi­cation reaction, which is combined with CRISPR-​​Cas-​​dependent amplicon recog­nition assays for the differ­en­ti­ation of relevant SARS-​​CoV-​​2 variants. This is achieved by using several SNP-​​specific, FnCas9-​​mediated amplicon binding assays in combi­nation with a simple lateral flow readout (Milenia® HybriDetect). The sum of the results allows conclu­sions about the present SARS-​​CoV-​​2 variant. The authors called their method RAY which stands for Rapid variant AssaY. [4]

Differ­en­ti­ation of SARS-​​CoV-​​2 Variants (WT and N501Y) using CRISPR-​​FnCas9 for Specific Amplicon Binding


RAY for differ­en­ti­ation of SARS-​​CoV-​​2 Variants of Concern.

Request a detailled protocol for RAY Detection on Bio​-Protocols​.org

Nested Nucleic Acid Ampli­fi­cation

Nested ampli­fi­cation is a two-​​stage DNA ampli­fi­cation strategy in which the product of the first reaction serves as a template for the second one. In the follow-​​up reaction(s) several pieces of infor­mation can be generated. However, this form of additional analysis does not sound very attractive at first, because connecting a second nucleic acid ampli­fi­cation reaction to a first can be time-​​consuming, expensive and imprac­tical. Never­theless we see potential for innovation in this type of technology. The primer extension PCR (PEXT-​​PCR) can serve as an inter­esting example. Here, in a subse­quent ampli­fi­cation, SNP-​​specific primer(s) are elongated in the 2nd PCR under strictly defined condi­tions, which result in specific reaction products that are detectable via Lateral Flow. By combining successive ampli­fi­cation reactions. Very sensitive and precise multiplex-​​test systems can be developed.

Learn more about PEXT-​​PCR-​​LFA

Collection of Relevant Multiplex NALFIA Publi­ca­tions

Multiplex Detection of … NALFIA TypeReference
Cyprinid-​​Herpesvirus-​​3 & IACDuplex PCR & Hybridization Loose & Breitbach et al., 2020 (1)
Cyprinid-​​Herpesvirus-​​3 & Carp Edema VirusDuplex RPA (nfo-​​dependent)Soliman & El-​​Matbouli, 2018 (2)
Two Targets SARS-​​CoV-​​2Duplex LAMP & HybridizationBhadra et al., 2021 (3)
SARS-​​CoV-​​2 VariantsPCR & CRISPR/​Cas-​​AssayKumar et al., 2021 (4)
Authen­tifi­cation Cordyceps & IACDuplex PCRWong et al., 2015 (5)
Plasmodium Genus & P. knowlesiDuplex RPA (nfo-​​dependent)Lai et al., 2018 (6)
HIV /​ SARS-​​CoV-​​2 (semi-​​quantitative)Competetive RPA & HybridizationMancuso et al., 2021 (7)
  1.  a b c : Loose, F. N., Breitbach, A., Bertalan, I., Rüster, D., Truyen, U., & Speck, S. (2020). Diagnostic validation of a rapid and field-​​applicable PCR-​​lateral flow test system for point-​​of-​​care detection of cyprinid herpesvirus 3 (CyHV-​​3). PloS one, 15(10), e0241420. https://​doi​.org/​10​.​1371​/​j​o​u​r​n​a​l​.​p​o​n​e​.​0241420 ()
  2.  : Soliman, H., & El-​​Matbouli, M. (2018). Rapid detection and differ­en­ti­ation of carp oedema virus and cyprinid herpes virus-​​3 in koi and common carp. Journal of fish diseases, 41(5), 761 – 772. https://​doi​.org/​10​.​1111​/​j​f​d​.​12774 ()
  3.  a b : Bhadra, S., Riedel, T. E., Lakhotia, S., Tran, N. D., & Ellington, A. D. (2021). High-​​Surety Isothermal Ampli­fi­cation and Detection of SARS-​​CoV-​​2. mSphere, 6(3), e00911-​​20. https://​doi​.org/​10​.​1128​/​m​S​p​h​e​r​e​.​00911 – 20 ()
  4.  : Kumar, M., Gulati, S., Ansari, A. H., Phutela, R., Acharya, S., Azhar, M., Murthy, J., Kathpalia, P., Kanakan, A., Maurya, R., Vasudevan, J. S., S, A., Pandey, R., Maiti, S., & Chakraborty, D. (2021). FnCas9-​​based CRISPR diagnostic for rapid and accurate detection of major SARS-​​CoV-​​2 variants on a paper strip. eLife, 10, e67130. https://​doi​.org/​10​.​7554​/​e​L​i​f​e​.​67130 ()
  5.  a b : Wong, Y. L., Wong, K. L., & Shaw, P. C. (2015). Rapid authen­ti­cation of Cordyceps by lateral flow dipstick. Journal of pharma­ceu­tical and biomedical analysis, 111, 306 – 310. https://​doi​.org/​10​.​1016​/​j​.​j​p​b​a​.​2015​.​04.003 ()
  6.  a b : Lai, M. Y., Ooi, C. H., & Lau, Y. L. (2018). Recom­binase Polymerase Ampli­fi­cation Combined with a Lateral Flow Strip for the Detection of Plasmodium knowlesi. The American journal of tropical medicine and hygiene, 98(3), 700 – 703. https://​doi​.org/​10​.​4269​/​a​j​t​mh.17 – 0738 ()
  7.  : Mancuso, C. P., Lu, Z. X., Qian, J., Boswell, S. A., & Springer, M. (2021). A Semi-​​Quantitative Isothermal Diagnostic Assay Utilizing Compet­itive Ampli­fi­cation. Analytical chemistry, 93(27), 9541 – 9548. https://​doi​.org/​10​.​1021​/​a​c​s​.​a​n​a​l​c​h​e​m​.​1​c​01576 ()