Nucleic Acid Lateral Flow Immunoassay (NALFIA)

Creative Diagnostics - Food & Feed Analysis

Nucleic Acid Lateral Flow Immunoassay (NALFIA)

Nucleic Acid Lateral Flow Immunoassay (NALFIA)

NALFIA (Nucleic Acid Lateral Flow Immunoassay) is a general device that combines lateral flow technology and immunoassay principle for detection of nucleic acid related products, such as PCR products, genetic amplicons. NALFIA was first developed for immunoassay as the point-of-care (POC) tests in molecular diagnosis in 2006 and attracted a lot of interest from researchers and developers in the public and private sectors. Under some conditions where the molecular biology laboratory infrastructure is basic or almost absent, NALFIA as a simple and easy-to-use POC product, is particularly demanded. Until today, an increasing number of nucleic acid later flow test has been applied in the domain of infectious disease (human, animal, and plant), food safety analysis, genotyping and environmental contamination monitoring.

Advantages of NALFIA

NALFIA's analytes rely on the primers labeled with small molecules for nucleic acid amplification of the target sequence. The analyte then captured by corresponding gold-labeled small molecule antibody on the test line of the test strip, and develop color to achieve the purpose of detection. It has great potential in the diagnostic market due to the following characteristics:

Sequence independent, both DNA/RNA can be amplified before detection
Easier to operate
Cost-effective than other nucleic acid detection methods

The NALFIA and the LFIA set-ups are usually designed for testing the presence or absence of pathogens in food, feed or the environment. However, different from traditional colloidal gold lateral flow immunoassay (LFIA), NALFIA can be used in such infections when antibodies are absent (early infection) or when antibodies persist after cure, both DNA and RNA sequence can be amplified before detection and then to be detected. Compare with the preparation of immunoassay reagents such as antigen and antibody, the specific primer or hybridisation sequence design is far easier and fast. On the other hand, NALFIA is a very cost-effective product compared to agarose gels, RT-PCR, LAMP (loop-mediated isothermal amplification) etc.

Capture Systems of NALFIA

Capture Systems of NALFIA

A number of systems are available to capture the nucleic acid sequence of interest onto the lateral flow membrane. This can be achieved by means of non-covalent interactions between antigen and antibody or biotin and avidin or probe hybridisation and their combinations: (A) a ds-amplicon was obtained as analyte with one strand labelled with biotin and the other strand labelled with, e.g., fluorescein isothiocyanate or digoxigenine, then biotin will bind to the avidin-labelled nanoparticles and the other tag will bind to the anti-tag antibody, and resulting in the coloured signal. (B) nanoparticle-labelled reporter probe and biotin-labelled immobilised capture probe via avidin, single-stranded amplicon (ss-amplicon) hybridises with complementary reporter and capture probes. (C) nanoparticle-labelled reporter probe and bovine serum albumin labelled capture probe, immobilised through passive adsorption, ss-amplicon hybridises with complementary reporter and capture probes. (D) nanoparticle-labelled reporter probe; capture probe is immobilised at the test line through passive adsorption, ss-amplicon hybridises with complementary reporter and capture probes. Different systems can satisfy the detection needs of multiple target analytes.

Assay Principle

NALFIA is typically composed of a conjugate pad, nitrocellulose membrane, test line (coated with capture antibody), control line (coated with species-specific antibodies) and absorbent pad. The nucleic acid sample should be prepared using two primers labeled with different reporter molecules.

Assay Principle

Fluorescent dyes (FITC/FAM, Cy5, Texas Red)
Small molecules (Biotin, Digoxigenine, DNP)

Take FAM and Biotin as example, after an amplification procedure, the amplicons are labeled with the two reporters. The amplicons bind first to the gold-labeled FAM-specific antibodies in the conjugate pad and form an amplicons-antibody-gold particle complex. Then the complex moves forward by capillary action and is captured by biotin-ligand molecules on the test line and generate a visible band over the time. The unbound gold-labeled FAM-specific antibodies migrates the control line and will be captured by species-specific antibodies. With prolonged incubation time, the formation of an intensely colored control band appears.

Prospect Application

Instead relies upon recognition of tagged primers by antibodies, NALFIA is theoretically independent of sequence and can be applied to the detection of multiple different amplicons. However, there is still a disadvantage: not only the volume of sample but also the amount of target analyte are decreased which directly decreased the sensitivity. Researchers continuously investigate the signal enhancement capacities of new materials and chemistry to increase the sensitivity of nucleic acid lateral flow test in detecting nucleic acid hybridisation events. In fact, many articles have already studied the application of NALFIA in detecting E.coli, BK Virus, Listeria spp. and Cronobacter spp. etc. and achieved good results. Some researchers are trying to apply the NALFIA to COVID-19 rapid testing. We do expect NALFIA to play a greater role in the future analysis and testing market.

Please visit our FOOD SAFETY LATERAL FLOW STRIPS DEVELOPMENT SERVICE to see more.

References:

  1. Martina Blaková, et al. "Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeria monocytogenes in food." European Food Research and Technology 229.6(2009):867-874.
  2. Huang, Yi Huei , et al. "Development of a Nucleic Acid Lateral Flow Immunoassay for the Detection of Human Polyomavirus BK." Diagnostics 10.6(2020):403.
  3. Philippe, and Büscher. "Nucleic acid lateral flow tests for molecular diagnosis: an update. "Expert opinion on medical diagnostics (2011).
  4. Petra, et al. "Molecular diagnosis of malaria in the field: development of a novel 1-step nucleic acid lateral flow immunoassay for the detection of all 4 human Plasmodium spp. and its evaluation in Mbita, Kenya." Diagnostic Microbiology & Infectious Disease (2008).
  5. Kellner, M.J., Koob, J.G., Gootenberg, J.S. et al. “SHERLOCK: nucleic acid detection with CRISPR nucleases”. Nat Protoc 14, 2986–3012 (2019).
  6. Seal J, Braven H, Wallace P. Point-of-care nucleic acid lateral-flow tests. IVD Technol 2006;12:41-51.
  7. Tobias, et al. "Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula." Food Control 109.C(2020):106952-106952.
  8. Mens, P. F. , et al. "Direct Blood PCR in Combination with Nucleic Acid Lateral Flow Immunoassay for Detection of Plasmodium Species in Settings Where Malaria Is Endemic." Journal of Clinical Microbiology 50.11(2012):3520-5.
  9. Wang, Kan , et al. "The Application of Lateral Flow Immunoassay in Point of Care Testing: A Review." Nano Biomedicine & Engineering 8.3(2016).
  10. Posthuma-Trumpie, Geertruida A. , J. Korf , and A. V. Amerongen . "Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey." Analytical and Bioanalytical Chemistry 393.2(2009):569-582.
  11. van Amerongen A, Koets M, In: van Amerongen A, Barug D, Lauwaars M (eds) Rapid methods for biological and chemical contaminants in food and feed. Wageningen Academic Publishers, Wageningen (2005), pp 105–126.
  12. Edwards, Katie A. , and A. J. Baeumner . "Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization." Analytical & Bioanalytical chemistry 386.5(2006):1335-1343.
  13. Posthuma-Trumpie, Geertruida A. , J. Korf , and A. V. Amerongen . "Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey." Analytical and Bioanalytical chemistry 393.2(2009):569-582.
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