LTC Monica O

1
Development, Comparison, and Use of Nucleic
Acid-Based Diagnostics to Detect Arboviruses in
the Field

• LTC Monica OGuinn
• and
• Dr. John Lee
• US Army Medical Research Institute of Infectious
  Disease, Fort Detrick, Frederick, Maryland

2
Development of Field RT-PCR
Goal Standardized / Simplified protocols for
field use with conventional, real-time PCR, and
wicking assay

• Alphaviruses tested - Una, Trocara, EEE,
  Sindbis,WEE, and VEE
• Flaviviruses tested - West Nile, JE, YF, SLE,
  Karshi, Ilheus and dengue
• Bunyaviruses tested- Rift Valley fever virus
  (RVFV), CCHF, and sand fly fever Naples,
  Sicilian, and Toscana
• Other Plasmodium falciparum, Plasmodium vivax
• Species differentiation ROK anopheles and
  Leishmania spp.

3
Development of Regional Diagnostic Detection Kits
Example The Middle
East
Other Pathogens
Alphaviruses
Bunyaviruses
Flaviviruses
Malaria Leishmania Plague Q-Fever
West Nile virus Tick-borne encephalitis Dengue
CCHF Sand fly fever Rift Valley
fever Hantaviral disease
Sindbis Chikungunya ONN
4
Overview
Confirmation
Other Assays
Pool Triturate
RNA
Extract
Sequence Analysis
TRIzol
DNAzol
DNA
cDNA
PCR
Ready-To-Go
Blast Search on GenBank
Detection
Remaining PCR Product
Phylogenetic Tree
PCR Clean Up
Sequencing
5
Arthropod Studies in the Lab
Animal Studies RT-PCR
Transmission ()
Ticks
Positive
Animal Studies RT-PCR
Mosquitoes
Transmission (-)
Negative
6
Laboratory MethodsRift Valley Fever Virus
Detection
7
RVFV Primer DesignUse of Phylogenetic
Comparisons
Mauritania 19987
Burkina Faso 1984
Senegal 1984-1993
Uganda 1955
8
Selection of Real-Time RVFV Primers and Probes

• Forward primers
• 227-CGAAAGAAGGCAAAGCAACT
• 233-AAGGCAAAGCAACTGTGGAG
• 231-AGAAGGCAAAGCAACTGTGG
• 241-GCAACTGTGGAGGCTCTCAT
• Reverse primers
• 388-CCACTCACTCAAGACGACCA
• 437-TGCATCATATGCCTTGGGTA
• 375-CCACTCACTCAAGACGACCA
• Probes
• 312-ATCACGAGTTGCTGCCGCCC
• 312B-ATCACGAGTCGCTGCAGCTT

Amplicon size 100-150 bp
9
Development of RVFV Primer Sets Field Testing
4
3
5
2
6
8
9
11
12
1
7
10
lane 2, RVF 1/2 primer set lane 4, RVF 227F/388R
primer set lane 6, RVF PS2F/1R primer set lane
8, RVF PS2F/925R primer set lane 10, RVS/RVA
primer set lane 12, BCS 82C/332V primer set
(negative control primer set)
10
Optimization Parameters for Assay Development

• RNA purification method
• Annealing time and temperature
• Magnesium titration
• Primer and probe titration
• Use of Probe vs SYBR Green

End Result Acceptable Limit of Detection
11
Optimization of Primer Annealing Temperature for
Detecting RVFV
70.0C 68.9C 66.8C 63.9C 60.3C 57.4C 55.2C 5
4.0C
RVS-RVA-RVP primer probe set
12
Optimization of Magnesium for Detection of RVFV
using SYBR green
No Additional Magnesium 25 nanomoles added 50
nanomoles added 75 nanomoles added 100 nanomoles
added 125 nanomoles added Negative control, no
virus
Performed on the RAPID real-time thermal cycler
13
Optimization of Primer Concentration for
Detecting RVFV
0.5 pmoles primer 1.0 pmoles primer 1.5 pmoles
primer 2.0 pmoles primer 2.5 pmoles primer 3.0
pmoles primer 3.5 pmoles primer 4.0 pmoles
primer 4.5 pmoles primer 5.0 pmoles primer 7.0
pmoles primer 10.0 pmoles primer Neg control (0.5
pmoles primer) Neg control (10 pmoles primer)
10 picomoles primers
3.5 pmoles
7 pmoles
2.5 pmoles
1.5 pmoles
Neg control (10 pmoles primer)
Performed using the RAPID real-time thermal
cycler using SYBR Green
14
Limits of Detection for RVFV Using RVS-RVA Primer
Undiluted cDNA
RVFV cDNA undiluted 15 dilution 125
dilution 1125 dilution 1625 dilution 13125
dilution 115,625 dilution 178,125
dilution 1390,625 dilution Neg CNTL, primers only
Performed using the RAPID real-time thermal
cycler and SYBR Green
15
RVFV Response JAN-FEB 2007
Field Methods
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KENYA
KENYA
RVFV Outbreak
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Setting Traps Near Houses
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Mosquito Identification and Pooling
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Processing of Specimens at BSL3 CDC Kenya
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Extraction of RNA from Ground Mosquito Pools
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Dipstick Testing of Positive Mosquito Pools
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RT-PCR in KEMRI PCR Hood
23
Loading Gels After the Completion of the PCR
24
Transitioning the Lab to the Field Rift Valley
Fever Virus Detection
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Field Gels Showing RVFV Positive Pools


Collected in Kurabull on 4 January 2007 Species
Ae. mcintoshi (sample 372) Product 551 bp,
Ibrahim et al. 1997
Collected in Kurabull on 4 January 2007 Species
Ae. Ochraceus (Sample 310) Product 551 bp,
Ibrahim et al. 1997
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Field Results Using Real-Time PCR to Detect RVFV
Using Taqman Probes
KLF-014 (4) KLF-085 (9) KLF-091 (15) KLF-112
(33) Negative Control
KLF-014 (4) KLF-085 (9) Negative Control
Primer set was RVF 588F-946RC with RVF 642 probe
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Blast Report for RVFV Isolated in Kenya 2007
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Kenya Team

• USAMRU-Kenya
• KEMRI
• CDC-Kenya
• WRAIR Biosystematics
• MIDRP
• GEIS

Opinions,?interpretations, conclusions, and
recommendations are those of the?author and are
not necessarily endorsed by the U.S. Army
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QUESTIONS?