This document describes a study that aimed to develop a fast and inexpensive method to test for viruses like yellow fever and dengue fever in developing countries without expensive machines. The method uses helicase dependent amplification of viral DNA combined with a smartphone fluorimeter to detect fluorescence, indicating the presence of amplified viral DNA. The study demonstrated that viral DNA could be successfully amplified within 30 minutes and detected using only a smartphone camera and free image analysis software, providing a promising new tool for rapid testing in resource-limited settings.

1. DNA Measurement Using a
Smart Phone Fluorimeter
By: Carissa Henriksen
Arizona State University

2. Research Question
 How can we easily and quickly test for viruses in
developing nations, without the use of expensive
machines?
 Diseases such as Yellow Fever and Dengue Infection are
devastating many countries in South America, Africa,
and Asia.
 There are over 200,000 cases of yellow fever every year,
with 30,000 deaths (Domingo)
 There are over 100 million cases of Dengue Infection
(Khawsak)
 These diseases desperately need a fast, easy indicator to
determine if a person has one of these diseases.

3. Collecting Data 1
 The use of helicase dependent amplification is important
in this experiment, because it can be completed fairly
quickly. It is also done in one temperature, unlike other
amplification processes, where the reaction needs to be
heated and cooled multiple times.

4. Collecting Data 2
 This was the basic template for the amplified mix used
throughout the experiment
 Reaction Mix
 14 microliters of distilled water
 2.5 microliters of 10x Annealing Buffer
 2 microliters of Sodium Chloride
 1 microliter of Magnesium Sulfate
 2 microliters of dNTP
 1 microliter each of forward and reverse primer
 2 microliters of enzyme mix
 2.5 microliters of Template DNA

5. Collecting Data 3
 The Reaction mix was placed under lamps producing 60
degrees Celsius for 30 minutes, then added to SYBR
Green mixture

6. SYBR Green Mixture
Two different SYBR Green mixtures were
used to allow the DNA to fluoresce
 Mix 1  Mix 2
 2.5 microliters 10,000x  5 microliters 10,000x
SYBR Green 1 SYBR Green 1
 10 milliliters of Trizma  10 milliliters of Trizma
Hydrochloric Acid, 1M Hydrochloric Acid, 1M
pH 8 pH 8

7. Analyzing the Data
 After calibrating, the 28 uL of the sample was added to a
drop on a 30 well slide coated with teflon with 132 uL of
SYBR Green
 A blue light is then shone through the middle of the drop
to highlight flourescence
 A picture is then taken on a smart phone (could be done
with any digital camera)
 The picture(s) are then uploaded to a computer to be
analyzed on ImageJ, a free Java-based image processing
program
 Note: It is not always necessary to analyze on ImageJ,
most of the results can be seen with the naked eye
qualitatively!

8. Drop to be analyzed
Setup

9. Comparison of Data
No DNA Amplified DNA

10. Analyzing Data on Image J
 An elongated oval is traced on the bottom of the drop,
and then the “measure” tool is used to measure area,
integrated density, and mean gray value
 The integrated density is directly proportional to the
amount of material in the system
 Product of area and Mean gray value
 The Mean Gray Value is the average gray value within
the selection

11. Other Trials Done in Experiment
 These trials were done to verify that the DNA is actually
amplifying
 Mix #9: Solution without DNA (2.5 Extra microliters of
water)
 Mix #10: Solution without Primers (2 Extra microliters of
water)
 Mix #11: Solution without Enzymes (2 extra microliters
of water)
 Mix #12: Broken pipettor used

12. Data
Name Area Integrated Density MGV
Water Sample 43,120.00 1.78*105 4.14
80:80 Calibration 43,120.00 1.03*106 23.99
132:28 Calibration 43,120.00 1.06*106 24.52
Mixture 8 (normal) 43,120.00 1.72*106 39.89
Mix 9 (no DNA) 43,120.00 1.42*106 32.91
Mix 10 (no Primers) 43,120.00 9.31*105 21.58
Mix 11 (no Enzymes) 43,120.00 6.20*105 14.38
Mix 12 (broken pipette) 43,120.00 4.44*105 10.29

13. Mean Integrated Density
1.80E+06 1.72E+06
1.60E+06 1.42E+06
1.40E+06
1.20E+06 1.06E+06
9.31E+05
1.00E+06 1.03E+06
8.00E+05 6.20E+05
6.00E+05 4.44E+05
4.00E+05
1.78E+05
2.00E+05
0.00E+00

14. Mean Gray Value
39.89
40
32.91
35
30
23.99 24.52
25 21.58
20
14.38
15 10.29
10
4.14
5
0

15. Results
 Results were calculated by taking the percentage of the
integrated density of each reaction divided by the
integrated density of the 132:28 calibration.
 Mix 12 Results: 41.9%
 Mix 11 Results: 58.6%
 Mix 10 Results: 88.034%
 Mix 9 Results: 134.25%
 Mix 8 Results: 162.7%

16. Discussion of Results
 The DNA was definitely amplified in Mix 8. In mix 9, the
results were so high because of possible contamination.
Mix 10 did not have the primers, which is essential to
actually amplify the DNA. Mix 11 did not have any
enzyme, which would not allow the reaction to be
completed in any reasonable time. Mix 12 just did not
work, resulting from a broken pipette used in the
measurements.

17. Conclusions
 In half an hour, the DNA was amplified.
 The system does not require any expensive machinery.
 Results are evident right away. No waiting!
 Another system is in the works to have multiple
reactions occur at once!

18. References
 Domingo, Christina, et al. "Advanced Yellow Fever Virus
Genome Detection in Point-of-Care Facilities and
Reference Laboratories." Journal of
Clinical Microbiology 50.12 (2012): 4054-60. PDF file.
 Khawsak, Phaisan, Sirichai Phantana, and Kosum
Chansiri. "Determination of Dengue Virus Serotypes in
Thailand Using PCR Based Method." Southeast Asian
J Trop Med Public Health 34.4 (2003): 781-85. PDF file.
Back to “Research Question” (slide 2)

19. Acknowledgements
 Dr. Antonio Garcia; Arizona State University- provided
research opportunity and lab
 Dr. Karmella Haynes; Arizona State University- ran gel
electrophoresis experiments and co-author
 Angle Lastra; co-author