Rapid Direct Detection of Dengue Virus in Blood Samples by Real Time-Polymerase Chain Reaction
(RT-PCR) using a Portable Point-of-Care Device
William M Nelson, Tracy Fecteau, Lisa Cockrell, Neeraja Venkateswaran, Kyle Armantrout, Tetracore, Inc. Rockville, MD.
ABSTRACT
Introduction: Dengue virus (DENV) is a mosquito borne human virus causing both asymptomatic and severe
DENV 2 Dilution Average
in Blood
Ct Value
infection in tropical and subtropical regions of the world. A rapid detection of the virus is critical to patient
Std Dev
% CV
management and for surveillance of disease. A dry format quantitative reverse transcriptase polymerase chain
reaction assay was developed for field use and systematically evaluated previously using Cepheid SmartCycler
1/10
21.7
0.538
2.48
platform for rapid detection of DENV1. In the current study we optimized this assay for use with T-COR 8™
1/100
25.1
0.446
1.78
integrated system that includes a portable thermocycler, simple sample collection and processing devices to
1/1000
27.7
0.546
1.98
1/10000
30
0.898
2.99
1/100000
30.9
1.521
4.93
detect DENV in whole blood samples directly. The objective of the study was to develop an assay suitable for not
only the laboratory settings but also for point-of-care settings, low resource settings and austere environments.
Methods: The assay specificity was previously tested by using a panel of related flaviviruses. Eighty one
confirmed dengue positive samples and 25 negative samples were tested to validate the assay on Cepheid
SmartCycler. For optimization of this assay on T-COR 8 integrated system we spiked cell culture derived viruses in
Figure 3: Comparison of Standard Curves for DENV2 on
Traditional Thermocycler ABI 7500 and Portable T-COR 8
normal human blood samples and then a titration was done. We also compared amplification of extracted RNA
from samples with testing of blood samples directly without RNA extraction, using a novel rapid plasma dilution
Table 3: DENV2 was diluted in whole blood
and tested on T-COR 8. Average Ct Value of
7 replicates of each dilution over 5 logs is
shown here.
buffer (RPDB). Use of RPDB with a simple collection device removed the need for centrifuge for plasma
separation, RNA extraction and several other steps that may use a pipette. Both finger prick and venous puncture
for blood collection were tested in this system. Comparison of this integrated system with traditional bench top
thermal cycler was performed using the spiked samples. Sample collection for further validation of this integrated
system is ongoing.
Results: Using the set of 81 confirmed positive and 25 negative samples the assay was found to be 98.77%
Figure 1: T-COR 8 portable thermocycler with Two User Friendly Screens (A) Tabular Display of the Results Data,
Color Coded Positive/Negative calls for each sample, Interface Buttons for 8 Individual Wells (B) Real Time
Graphic Display of Results Data
sensitive and 100 % specific on Cepheid SmartCycler. Titration of spiked samples showed a wide dynamic range
with 5 logs of linearity when T-COR 8 integrated system was used. Comparison of tests using extracted samples
and direct testing from blood samples using novel RPDB buffer show similar results. Test performed on T-COR 8
integrated system gave similar or better results than a traditional bench top thermal cycler.
Conclusions: The data from this study shows that using this novel assay system presence of DENV can be
detected rapidly in blood samples without the need for RNA extraction and other traditional lab supplies. This
novel system can be used equally effectively in labs, in field, in austere environments, on nursing cart or at the
bed side for detection of DENV. This system includes T-COR 8 device, simple sample collection and processing
Figure 4: Comparison of Amplification Curves for DENV
2 RNA from Spiked Blood Samples with Extraction and
No Extraction
system with the dried down test reagents for DENV, and software that provides test results both locally and
remotely.
Results and Discussion
INTRODUCTION
Dengue has re-emerged worldwide due to urbanization, increase in travel and climate change, becoming a major
global public health concern. Various methods used currently for laboratory detection of DENV infection include
Figure 2: Disposable Simple Device for Blood Sample Preparation and C2T cartridge for Nucleic
Acid Amplification without Extraction
virus culture, viral antigen detection, immunohistochemistry, serological methods, and viral RNA detection.
antigens such as NS1 is not very sensitive. Serological methods, in which antibodies to DENV are detected, may
not be very specific due to cross reaction with other flaviviruses making interpretation of results difficult and are
3
.
Virus isolation and culture from patient blood samples is
very cumbersome and time consuming, requiring expertise and an appropriate laboratory facility. Although there
is a lack of availability of specific antiviral drugs for treatment of these diseases yet timely intervention can avoid
development of more severe symptoms like Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome
The PAN DENV assay was previously validated using Cepheid SmartCycler. SmartCycler has been considered as
closest to a portable system traditionally. The assay was found to be 98.77% sensitive and 100% specific when 81
confirmed positive and 25 negative samples were tested1. In this study we evaluated T-COR 8 portable
However, there are many challenges to these dengue detection and diagnostic methods. Detection of DENV
not very sensitive for detection of acute infections2,
Figure 5: Amplification Curves showing
Repeatability of Assay with 4 Replicates in 4
Independent Wells on T-COR 8
Feature
Description
Networkable via WiFi/Ethernet for remote access and data
download
Independently programmable overcoming the limitation of
batch processing
“Cloud” Ready
8 independent wells
thermocycler for DENV detection in spiked blood samples. Various hardware and software features for T-COR 8
thermocycler are described in Table 1 and Table 2 respectively. Efficiency of PCR by integrated sample preparation
and amplification on T-COR 8 instrument was compared with traditional methods on lab based thermocycler ABI
7500. A comparison of standard curves obtained from dilution of DENV 2 in TE buffer and Ct Values obtained from T
-COR 8 portable thermocycler were compared with ABI 7500. Efficiency of PCR reaction on T-COR 8 over a 5 log
Multiple protocols simultaneously
8 independent wells can run 8 different protocols
Multiplex capable
Currently 4 channels available for FAM, DFO, CY5 and Texas
Red, possibility to expand up to 6 colors
the curve for T-COR 8 is -3.44 as opposed to that obtained on ABI 7500 at –4.56 (Figure 3). DENV 2 Spiked blood
(DSS). Thus, a significant need exists for the development of diagnostic methods that can be used in low
range of dilution was found to be better than ABI 7500 for the reaction DENV 2 mix used in this test. The slope of
samples were processed through the blood collection device and tested on T-COR 3 using C2T cartridge. Traditional
resource settings and with minimal training at the point-of-care. This study was designed to develop a fully
Touch Screen
10.4 Inch (26.4cm) with Virtual Keyboard
RNA extraction was also done with each dilution. The spiked samples were also tested directly without sample
integrated system for rapid and direct detection of DENV in whole blood that can be used equally effectively in
Battery operated
4 Hours of Average Thermocycling Battery life
processing or RNA extraction. A comparison of amplification curves from T- COR 8 for this study is shown in Figure
laboratories and in low resource settings.
Light weight
Less than 4.5Kg
4. Ct values obtained two dilutions tested for novel sample processing and traditional RNA extraction methods are
Integrated Bar code reader
Linear or two dimensional
comparable. There is no pipetting or centrifugation required for novel sample processing method making it very
Materials and Methods
simple and rapid for use with minimal training and equipment requirements. At least four replicates of each of the 5
Table 1: Salient features of Tetracore T-COR 8 Hardware
log dilutions of DENV 2 in blood were tested by 2 performers. Table 2 shows that method was very reproducible
To establish the equivalence of a traditional thermocycler with our portable thermocycler T-COR 8 we used 10 fold
serial dilutions of DENV 2 procured from ZeptoMetrix in TE buffer. Testing of these samples was done using a PAN
assay that targets conserved sequences for all four serotypes of DENV with two forward and two reverse primers.
Probe is FAM (fluorescein) labeled and BHQ is used as quencher. For internal Control (IC) one forward and one
reverse primer were used and probe is labeled with ATTO647N and quencher is BHQ. The Ct values were
generated by the Smart CT™ analysis software on T-COR 8 (Figure 1).
For optimizing the simple protocol for detection of DENV in whole blood without RNA extraction, DENV 2 virus was
spiked in whole blood at different levels. For optimization of sample preparation, processing of samples was done
with a coefficient of variance (%CV) below 5% for Ct values. A higher %CV was seen at the higher dilution as
Feature
Description
Software written in HTML 5 and is platform independent and
viewable in any browser
Auto analysis of results with included Smart CT™ Software that
calls Positive/Negative
Conclusions
User friendly interface
Custom protocol creator
resource settings, the diagnostics industry has not prioritized this need. Major limitation of RT-PCR based assays
Individual well control
View data from individual wells and channels
Versatile Software
Analysis Algorithm
Data storage
Data stored locally via SD card
novel simple sample preparation with no extraction was compared with traditional RNA extraction method. The
Report Accessibility
Download report via network or USB thumb Drive
Real time data display
View data in real time graphical or table format
method was further validated by two different operators using various spike levels and running at least 4
replicates per spike level.
Despite the lack of much needed diagnostic tools that will be very useful for rapid detection of DENV in low
being the need for extraction of RNA from samples requiring accurate pipetting, centrifugation and other basic skills
for a good quality RNA template generation. In this study we have shown that a simple sample processing protocol
using a novel buffer formulated for isolation of plasma using a simple dropper like device shown in Figure 2. The
C2T cartridge (Figure 2) was used for simultaneous template preparation and nucleic acid amplification. The novel
expected. The amplification curves for 4 replicates at limit of detection of 1/100000 dilution are shown in Figure 5.
integrated with nucleic acid amplification using a portable thermocycler can generate quality template for DENV 2
from blood samples that can be effectively detected with our portable thermocycler T-COR 8.
Further
investigations are being done to evaluate this system with more serotypes of DENV.
References:
Table 2: Salient features of Tetracore T-COR 8 Software
1. Wu Sj, Pal S et al (2008) A dry-format field-deployable quantitative reverse transcriptase polymerase chain reaction assay for diagnosis of dengue infection. Am J Trop.
Med. Hyg., 79 (4), 505-510.
2. Peeling RW, Artsob H, et al (2010) Evaluation of diagnostic tests: dengue, Nature Reviews Microbiology , S30-S37
3. Basile AJ, Horiuchi K, Panella AJ, Laven J, Kosoy O, et al (2013) Multiplex Microsphere Immunoassays for the Detection of IgM and IgG to Arboviral Diseases. PLoS ONE 8
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30th Annual Clinical Virology Symposium, Daytona Beach, Florida, April 27th-30th 2014