1. Development of a Pan-Dengue Diagnosis Platform Using Direct Serum Samples
Zhuoyu Ni¹†, Regina Heineman¹, Theron Gilliland, Jr.², Allison Dauner², Subhamoy Pal², Robert D. Hontz3, Shuenn-Jue Wu²
1Agave BioSystems, Ithaca, NY
²Naval Medical Research Center, Silver Spring, MD
3U.S. Naval Medical Research Unit No. 6, Lima, Peru
†Corresponding author: Zhuoyu Ni
Background
Dengue virus (DENV) is transmitted by the Aedes mosquito vector in the tropical areas
DENV causes dengue fever with symptoms ranging from mild to severe, accompanied by debilitating and potentially fatal outcomes
No vaccine treatment is available for Dengue diseases
Early diagnosis is critical for supportive patient care and disease management
A pan-Dengue diagnosis platform has been developed combining a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and a companion portable prototype
RT-LAMP vs. qRT-PCR
Application of RT-LAMP in DENV RNA detection
RT-LAMP is a method for specific, rapid, and robust amplification of RNA targets
LAMP is less susceptible to inhibitors in blood samples than regular PCR
RT-LAMP instrument requires less user training to operate
Time (minutes)
Fluorescence (Counts)
RT-LAMP
qRT-PCR
Sample Preparation
Serum used directly in assay
<1 minute
Viral RNA isolation from serum required
30-60 minutes
Assay set-up and operation
Mix serum and reagent,
60 minute reaction or less
Add reagent to purified RNA,
Instrumentation
Heat block for isothermal reaction
Thermal cycling sample holder
Readout
Real-time fluorescence optics,
others
Real-time fluorescence optics
Software
Time to threshold fluorescence
Ct value: number of cycles to fluorescence threshold
Real-time monitoring of fluorescence from RT-LAMP reactions.
“Threshold for positivity” (red dashed line) – 10 SD above the background fluorescence
“Time to positivity” (minutes) – the time when reaction fluorescence increases above that threshold.
AMV Reverse Transcriptase
Bst Polymerase 1 2 3
RT-LAMP Primer Design and Test
Serum vs. Whole Blood as Input
Impact of serum on RT-LAMP
Prototype Design and Development
Serum
Blood
RNA Purification
No Sample Prep
DENV3
DENV4
Mock Media
Virus Media
Time to Positivity (minutes)
Time to Positivity (minutes)
Time to Positivity (minutes)
Time to Positivity (minutes)
Illustration of target sequences and relative positions of RT-LAMP primers.
(Modified from Nat Protocol 2008;3(5): )
Serotype-specific primers are color-coded as on the left.
Sizes of dots indicate relative concentrations in the mix.
DENV 3 Viral RNA copies per reaction
Virus media – DENV-infected C6/36 cell media
Spiked human serum or blood – 10% virus media or mock media
In each 25 µl of serotype-specific RT-LAMP reaction, 0.5 µl of serum/blood was used.
Every reaction contains 1 μl of serum or equivalent amount of purified RNA.
The time-to-positivity for no-template controls are over the 90-min reaction time (not plotted).
All reactions contained 100 copies of viral RNA.
The time-to-positivity for no-template controls are over the 90-min reaction time (not plotted).
Portable and enclosed prototype
Touchscreen software operation
Temperature controlled sample holder for 8 well strip tubes
Real-time fluorescence data display
Readout results immediately at the conclusion of the reaction
Serum slightly increases the time to positivity
Susceptibility to the amount of serum in the reaction varies with serotype-specific reactions.
Differential amplification of positive and negative samples varies with serotype.
The RT-LAMP reaction condition is more tolerant to serum than blood samples.
Multiple primer mixes were designed to amplify the conserved 3’-UTR region of DENV viral RNAs.
“B” set of pan-Dengue primers was selected for best discrimination of positive (all 4 DENV serotypes) and negative samples. 4 5 6 7
Mg2+ optimization of RT-LAMP Assay on Prototype
RT-LAMP Prototype Performance
RT-LAMP Prototype Story Board
Buffer Optimization to Improve Serum Tolerance
Control Zone
Water in Tube
Replicates test on the 8-tube strip
Fluorescein (µM)
Fluorescence (Counts)
Each reaction contains 0.5 µl of serum.
Percentile of Agreement is calculated from comparison of RT-LAMP results to qRT-PCR standards.
Condition A – Buffer A containing 7 mM MgSO4, and 4 µl of serum sample
Condition B – Buffer B containing 8 mM MgSO4, and 2 µl of serum sample
Condition C – Buffer C containing 7 mM MgSO4, and 0.5 µl of serum sample
Percentile of agreement comparing RT-LAMP to qRT-PCR results is shown.
Increasing [Mg2+] can improve sensitivity, but may cause false positives
7mM Mg2+ improves detection sensitivity and slightly compromise the detection specificity.
DENV-2 , -3, -4 vs. Negative (Duplicates)
Fluorescence (Counts)
Time (minutes)
Negative Ctrl
DENV-2
DENV-3
DENV-4
A 5-min preheat is included before each LAMP reaction.
Minimal variation in fluorescence detection among the 8 tubes.
Duplicate results are very similar.
Limit of Detection (LOD) is 10^4 RNA copies per ml serum.
Condition B gives best analytical specificity and sensitivity.
Condition B was selected for the clinical test. 9 10 11 8
Clinical Sample Test
Clinical Test Results
Pan-Dengue Diagnosis Platform
Acknowledgements
We thank all the personnel at Agave BioSystems for their support and discussions. We would also like to thank Mr. Steev Loyola at US NAMRU-6 for helping with the clinical samples and independent tests. Acknowledgements also go to Dr. Timothy Endy (Upstate Medical University) for providing the Dengue viruses; and to Nanohmics for help with prototype development. This work was supported by the SBIR award to Agave BioSystems sponsored by U.S. Army Medical Research and Material Command (W81XWH-10-C-0038).
Key Features
Minimal sample preparation: serum dilution
Pan-Dengue assay : detect all 4 serotypes of Dengue RNA in single-tube isothermal reactions
Portable device: enclosed instrument with 8-sample capacity, thermal control, real-time optical monitoring, interactive user interface, instant readout
Rapid assay: complete within 60 minutes
Accurate detection: both clinical sensitivity and specificity at 96.7%
Limit of detection: detect 104 RNA copy/ml serum from 2 µl of serum sample
POC adaptability: isothermal reaction, minimal sample processing, and POC devices are adaptable to molecular diagnosis of many diseases
Clinical Serum Samples:
From patients with acute Dengue infection (US NAMRU-6, Peru)
Including 30 negatives, 8 of each DENV serotype except 6 for DENV-3
Independently verified by conventional RT-PCR and gel electrophoresis (NAMRU-6)
Corresponding RNA samples were provided for measuring DENV RNA copy numbers by qRT-PCR after the RT-LAMP test.
RT-LAMP Test Conditions:
Serum samples were blinded by staff at NMRC (Silver Spring, MD).
Each 25 µl reaction contains 2 µl serum sample (Condition B).
Each sample was tested in duplicates, one on each of the prototypes.
Reaction was performed at 60°C with 5 minute pre-heat time and the cut-off for time-to-positivity was 50 minutes.
“Positive” (+) – both replicates cross the diagnostic threshold in ≤ 50 min
“Negative” (-) – neither replicates cross the threshold in ≤ 50 min
“Equivocal” (+/-) – two replicates show opposite trend
Conventional RT-PCR was used as analytical gold standards performed independently at US NAMRU-6 lab (Lanciotti, et al., (1992) J Clin Microbiol. 30(3):545-51).
Notes
The views expressed in this poster are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. The study protocol was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable Federal regulations government the protection of human subjects. This work was performed when Mr. Theron Gilliland, Jr., Dr. Allison Dauner, and Dr. Subhamoy Pal were employed by the Henry M. Jackson Foundation for the Advancement of Military Medicine; and Dr. Shuenn-Jue Wu and LT Robert D. Hontz were employed by the United States Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that 'Copyright protection under this title is not available for any work of the United States Government.' Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as a part of that person's official duties.
Future Improvements
Multi-test format: currently for Dengue and Q-Fever, adaptable for other tropical diseases.
Portability: miniaturization
Capacity: 96-well format
Point-of-Care capability: reagents can be lyophilized for long-term storage in resource-limited settings
The pan-Dengue assay has clinical specificity and sensitivity both at 96.7%.
The DENV RNA copy numbers in the acute samples range from 7.57e+3 to 2.14 e+8 per ml serum (data not shown). 12 13 14