# Multiplex Detection of Respiratory Pathogens Using Due Bio LF-detect 3-Plex Lateral Flow Strips
## Abstract
Single-target diagnostic tests limit throughput and increase sample consumption in respiratory pathogen screening. Due Bio’s LF-detect 3-Plex Nucleic Acid Lateral Flow Strips enable simultaneous detection of three distinct targets in a single reaction—reducing costs and accelerating results for POCT manufacturers and clinical laboratories. This application note demonstrates a streamlined workflow for multiplexed detection of influenza A, influenza B, and SARS-CoV-2 using Due Bio’s proprietary triple-line capture technology.
## Introduction
Respiratory infectious diseases pose significant diagnostic challenges, particularly during seasonal outbreaks when multiple pathogens circulate simultaneously. Traditional single-plex assays require separate reactions for each target, consuming precious samples and reagents while delaying clinical decisions.
Multiplex detection addresses these limitations by enabling concurrent analysis of multiple targets from a single specimen. Due Bio’s LF-detect 3-Plex lateral flow strips incorporate three spatially separated test lines, each capturing a distinct reporter molecule corresponding to a specific pathogen target. This platform is ideal for syndromic testing panels, respiratory virus screening, and co-infection monitoring.
As a leading POCT manufacturer, Due Bio continues to innovate multiplex solutions that balance sensitivity, specificity, and ease-of-use for point-of-care deployment.
## Materials Required
– **Due Bio LF-detect: 3-Plex Nucleic Acid Lateral Flow Strips** (Cat. No. JY0303)
– **Due Bio IVD Reagents** (Multiplex PCR or RPA master mix)
– Target-specific primer sets (Influenza A, Influenza B, SARS-CoV-2)
– Dual-labeled reporters (Biotin-FAM, Biotin-HEX, Biotin-Cy5)
– RNA extraction kit or rapid lysis buffer
– Thermal cycler or isothermal amplification device
– Micropipettes and filtered tips
– Timer or stopwatch
*Note: All Due Bio LF-detect strips are supplied ready-to-use (50 strips/tube) in moisture-proof aluminum foil packaging. Store at 4–30°C in a dark, dry place. Shelf life: 12 months.*
## Step-by-Step Protocol
### 1. RNA Extraction
Extract total RNA from nasopharyngeal swabs using your preferred method. For rapid testing, use Due Bio’s 5× Lightning Nucleic Acid Release Reagent (5-minute room temperature lysis). Ensure final RNA concentration is between 1–100 ng/μL for optimal amplification.
### 2. Multiplex Amplification
Prepare a 25 μL reaction containing:
– 12.5 μL 2× Multiplex master mix (Due Bio IVD Reagents)
– Forward/reverse primers for all three targets (0.4 μM each)
– Three distinct dual-labeled reporters (0.2 μM each)
– Extracted RNA template (5 μL)
– Nuclease-free water to final volume
Run amplification: 50°C (15 min reverse transcription), 95°C (3 min polymerase activation), then 40 cycles of 95°C (15 sec) / 60°C (1 min). For isothermal methods (RPA/RAA), incubate at 37–42°C for 20 minutes.
### 3. Strip Application
Remove one 3-Plex strip from the tube. Immediately reseal the tube cap tightly to protect remaining strips from moisture. Add 80–100 μL of amplified product directly to the sample well. No additional buffer or running buffer is required.
### 4. Result Interpretation
Read results at 15 minutes under normal lighting conditions:
– **Control Line (C)**: Must appear for valid test. Indicates proper fluid flow and strip integrity.
– **Test Line 1 (T1)**: Influenza A positive (captures FAM-labeled reporter)
– **Test Line 2 (T2)**: Influenza B positive (captures HEX-labeled reporter)
– **Test Line 3 (T3)**: SARS-CoV-2 positive (captures Cy5-labeled reporter)
Any combination of test lines may appear depending on infection status. Single, dual, or triple positive results are all valid interpretations.
### 5. Documentation and Disposal
Photograph results within 30 minutes for record-keeping. Dispose of used strips and reaction tubes according to local biohazard regulations. Do not interpret results after 30 minutes, as background signal may increase.
## Troubleshooting
### Q: One or more test lines are missing in positive controls.
**A**: This indicates primer competition or imbalanced reporter concentrations. Optimize primer concentrations individually first, then re-titrate in multiplex format. Ensure all three reporters are labeled with distinct fluorophores compatible with strip capture antibodies. Due Bio technical support can provide optimized primer/probe concentrations for common respiratory panels.
### Q: Background signal appears between test lines.
**A**: Excessive amplification product or insufficient washing can cause background. Reduce cycle number by 2–3 cycles or dilute amplified product 1:5 with nuclease-free water before strip application. Ensure strips are stored properly and not exposed to humidity.
### Q: Control line is weak or absent.
**A**: Insufficient sample volume or degraded reagents may cause control line failure. Ensure at least 80 μL of reaction mixture is applied. Verify that amplification was successful by running an agarose gel. If problems persist, try a new strip from a freshly opened tube.
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## Conclusion & OEM/ODM Call-to-Action
Due Bio’s LF-detect 3-Plex strips streamline multiplex pathogen detection without complex instrumentation. The platform supports up to three simultaneous targets with clear, visually interpretable results in 15 minutes.
**Established in 1987**, Due Bio is a global POCT manufacturer committed to advancing accessible molecular diagnostics. Beyond our standard LF-detect product line, we provide comprehensive **IVD Reagents OEM/ODM services** for partners worldwide—including custom multiplex panel design, private labeling, and large-scale manufacturing under ISO 13485 quality systems.
Whether you’re developing a respiratory virus panel, a gastrointestinal pathogen assay, or a custom syndromic test, Due Bio’s technical team can support your project from prototype 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.*