# Field-Deployable Pathogen Detection by Combining LAMP Amplification with Due Bio LF-detect Strips
## Abstract
Isothermal amplification methods like LAMP (Loop-mediated Isothermal Amplification) offer rapid, equipment-free nucleic acid detection—but visual interpretation of turbidity or color change can be subjective and error-prone. Due Bio’s LF-detect Universal lateral flow strips provide objective, binary readouts for LAMP assays with 100-fold improved sensitivity over naked-eye detection. This application note presents a validated workflow for combining LAMP with lateral flow detection for field-deployable pathogen testing.
## Introduction
LAMP amplification has gained widespread adoption for point-of-care molecular diagnostics due to its isothermal nature (60–65°C), rapid turnaround (15–30 minutes), and tolerance to inhibitors. However, conventional LAMP readout methods—turbidity, colorimetric dyes, or intercalating fluorescence—suffer from subjectivity, false positives, or equipment requirements.
Lateral flow detection addresses these limitations by providing a simple, visual, and unambiguous yes/no result. Due Bio’s LF-detect strips capture biotin- and FAM-labeled LAMP amplicons via a double-antibody sandwich mechanism, producing a visible test line within 15 minutes.
As a POCT manufacturer since 1987, Due Bio has optimized this combined workflow for maximum sensitivity, specificity, and field robustness across diverse pathogen targets.
## Materials Required
– **Due Bio LF-detect Universal Lateral Flow Strips** (Cat. No. JY0201)
– **Due Bio IVD Reagents** (LAMP master mix, Bst 2.0 or Bst 3.0 polymerase)
– Target-specific LAMP primers (F3, B3, FIP, BIP, optionally FLoop, BLoop)
– 5′-Biotin-labeled FIP primer and 5′-FAM-labeled BIP primer
– Sample lysate (prepared with Due Bio Lightning Reagents)
– Heat block, water bath, or portable LAMP heater (60–65°C)
– Micropipettes (10 μL, 100 μL) and filtered tips
– Timer or stopwatch
– Nuclease-free water for dilution
*Storage: LF-detect strips at 18–28°C (protected from moisture); LAMP reagents at –20°C. Shelf life: 12 months.*
## Step-by-Step Protocol
### 1. LAMP Primer Design
Design 4–6 LAMP primers targeting your pathogen of interest using standard tools (PrimerExplorer V5, LAMP Designer, or NEB LAMP Primer Design Tool). Target regions should be 100–200 bp with high conservation across strains. Label the FIP primer with 5′-Biotin and the BIP primer with 5′-FAM. Typical primer concentrations: F3/B3 (0.2 μM each), FIP/BIP (1.6 μM each), FLoop/BLoop (0.8 μM each if used).
### 2. LAMP Reaction Setup
Prepare a 25 μL reaction containing:
– 12.5 μL 2× LAMP master mix (Due Bio IVD Reagents, contains dNTPs, MgSO4, betaine, buffer)
– Bst 2.0 or Bst 3.0 DNA polymerase (8 U)
– Primer mix (final concentrations as above)
– Sample lysate (2 μL, prepared with Due Bio Lightning Reagent)
– Nuclease-free water to final volume
Include a no-template control (NTC) with water instead of sample, and a positive control with known target DNA in each run.
### 3. Isothermal Amplification
Incubate at 63°C for 20–30 minutes in a heat block, water bath, or portable LAMP heater. For Bst 3.0 polymerase, 65°C for 15 minutes may suffice due to enhanced strand displacement activity. Optionally inactivate polymerase at 80°C for 5 minutes to reduce carryover contamination risk.
### 4. Lateral Flow Detection
Remove one LF-detect Universal strip from the tube. Reseal the tube cap tightly immediately to protect remaining strips from moisture. Dilute amplified product 1:5 with nuclease-free water (optional, reduces background signal). Add 80–100 μL of diluted (or undiluted) amplified product to the sample well. Wait 15 minutes at room temperature.
### 5. Result Interpretation
Read results under normal lighting conditions at 15 minutes:
– **Positive**: Both control (C) and test (T) lines visible—biotin/FAM-labeled amplicons are captured by anti-FAM antibody at test line
– **Negative**: Control line (C) only—no amplicons present, only excess primers flow through
– **Invalid**: No control line—insufficient sample volume or strip degradation; repeat with new strip
Photograph results within 30 minutes for documentation.
## Troubleshooting
### Q: Test line is faint or absent in positive samples.
**A**: Verify primer labeling efficiency (>80% for both Biotin and FAM) by ordering from reputable suppliers. Increase LAMP incubation time to 30 minutes. Ensure strips are not expired or exposed to humidity—check desiccant in tube. Try concentrating amplicons by ethanol precipitation if yield is low.
### Q: False positives in no-template controls.
**A**: LAMP is prone to carryover contamination due to high amplicon concentrations. Use separate pre- and post-amplification areas with dedicated pipettes. Include dUTP/UDG system in master mix to degrade carryover amplicons. Dilute amplified product 1:10 before strip application. Always include NTC in each run.
### Q: High background or smeared test line.
**A**: Excessive amplicon concentration can cause background. Dilute product 1:10 or 1:20 before strip application. Ensure proper washing during strip manufacturing (contact Due Bio if batch-specific issue). Store strips properly and use before expiration.
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## Conclusion & OEM/ODM Call-to-Action
Combining LAMP amplification with Due Bio LF-detect strips creates a field-deployable molecular diagnostic platform with objective, visual readout. The workflow requires only a heat source (60–65°C) and delivers results in under 45 minutes from sample to answer.
**Established in 1987**, Due Bio is a global POCT manufacturer committed to advancing accessible molecular diagnostics. Beyond our standard products, we provide comprehensive **IVD Reagents OEM/ODM services** including custom LAMP primer design, master mix optimization, strip customization, and regulatory support for CE/FDA submission.
Whether you’re developing an infectious disease test, a veterinary diagnostic assay, or a food safety screening kit, Due Bio’s technical team can support your project from feasibility to commercialization.
**Contact us today**: medtiger@foxmail.com
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*For research use only. Not for diagnostic procedures unless validated and approved according to local regulations.*