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Dispatch
Detecting Biological Warfare
Agents
Linan Song,* Soohyoun Ahn,* and David R. Walt*
*Tufts University, Medford, Massachusetts, USA
Appendix
Methods
Single Polymerase Chain Reaction
(PCR)
A PCR Master Mix from Promega Corporation (Madison, WI, USA) was applied
in all PCR assays. Asymmetric PCR was performed with a reaction mixture
containing 2.5 U Taq DNA polymerase, a 200-μmol/L concentration of
each deoxynucleoside triphosphate (dATP, dGTP, dCTP, and dTTP), 1.5 mmol/L
MgCl2 in 1´ reaction buffer, 500 nmol/L Cy3 modified
reverse primer, 50 nmol/L forward primer, and 10 μL DNA template from
the autoclaved bacterial cultures. The amplification profile included
an initial step at 95°C for 5 min, followed by 5 cycles of 95°C
for 1 min, 56°C for 1 min, and 72°C for 1 min; 30 cycles of 95°C
for 1 min, 54°C for 1 min, and 72°C for 1 min; and a final extension
step at 72°C for 7 min. PCR controls were run with the reaction containing
all the reagents for PCR amplification except template DNA.
Multiplex PCR
Primer pool I contained 7 primer pairs, BA2, BTK1, FT1, YP1, CB1, VA1,
and BM1. Primer pool II contained BA4, BTK2, FT2, YP2, CB2, VA2, and BM2.
Each primer pool has 1 primer pair for each biological warfare agent of
interest. Multiplex PCR was run as follows: an initial step at 95°C
for 5 min, followed by 5 cycles of 95°C for 1 min, 56°C for 1
min, and 72°C for 1 min; 40 cycles of 95°C for 1 min, 54°C
for 1 min, and 72°C for 1 min; and a final extension step at 72°C
for 7 min. PCR products were analyzed on 4% precast agarose gels (Invitrogen
Corporation, Carlsbad, CA, USA) and visualized on Gel Doc XR System (Bio-Rad
Laboratories, Hercules, CA, USA).
PCR products were denatured at 95°C for 5 min and chilled on ice.
The denatured PCR products (25 μL) were combined with 10 μL hybridization
buffer (2 mol/L NaCl and 0.2 mol/L phosphate-buffered saline [PBS]) and
hybridized to the multiplexed array for 30 min at room temperature. Tris-EDTA
(TE) buffer was used to wash the array after hybridization to remove the
unbound target DNA, and 20% formamide in PBS (50°C) was applied for
1 min to minimize nonspecific binding of target DNA to probes followed
by washing with TE buffer for another 1 min.
|
Appendix Table 1. Sequence
information of probes and primer pairs* (Download
PDF  [47
KB, 1 page])
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|
|
Organism
|
Target gene
|
Accession no.
|
Probe sequences and primer sequences
(5’-3’)
|
PCR product (bp)
|
Name
|
|
|
Bacillus anthracis
|
capA
|
M24150
|
P: 5´-CCGAGTCCTAGACAGGAAGCCTTAGCAAAAGCAATGGTTGATGCAGGGGC-3´
F: 5´-GGGGGGAAGAATACGATAAT-3´; R: 5´-GTTCTTGTCCATCCTTGGTC-3’
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188
|
BA1
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P: 5´-ACGCACTGGGGGGAAGAATACGATAATAAACCGAGTCCTAGACAGGAAGC-3´
F: 5´-TAGTAAATACGCACTGGGTT-3´; R: 5’-CATCCTTGGTCAAACACAAA-3´
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194
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BA2
|
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pagA
|
M22589
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P: 5´-GGAAGAGTGAGGGTGGATACAGGCTCGAACTGGAGTGAAGTGTTACCGCA-3´
F: 5´-AAAATGGAAGAGTGAGGGTG-3´; R: 5´-CCGCCTTTCTACCAGATTTA-3´
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120
|
BA3
|
|
P: 5´-TGTATCACCAGAGGCAAGACACCCCCTTGTGGCAGCTTATCCGATTGTAC-3´
F: 5´-TGTATCACCAGAGGCAAGAC-3´; R: 5´-TTCTGTGTGGATTGATCCTC-3´
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106
|
BA4
|
|
SASP-B
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AF359938
|
P: 5´-GAAGCTAAGAAAGCGCAAGCTTCTGGTGCTAGATTCAAAGCACAAATGC-3´
F: 5´-GTAAAACAAGCAAACGCACA-3´; R: 5´-GATTGTGATTGTTTTGCAGC-3´
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162
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BA5
|
|
P: 5´-CAAGCAGTAAAACAAGCAAACGCACAATCAGAAGCTAAGAAAGCGCAAGC-3´
F: 5´-TTTGCGACTGAAACAAATGT-3´; R: 5´-ATGCACGTCTGTTTCAGTTG-3´
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142
|
BA6
|
|
Brucella melitensis
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Sequence between BMEI1658 and BMEI1659†
|
AE009601
|
P: 5´-AGGTGTCATTAGGCTTGCTTGTGGACGAGAGAACGGTGGACCAAGGGAGA-3´
F: 5´-TCCGAGGGTAAACACCTAAG-3´; R: 5´-ACTCACGGACAGCATTAGGT-3´
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181
|
BM1
|
|
P: 5´-CTCTCACGGTCCCCATAATGGAGAGTGATCCGAGGGTAAACACCTAAGGG-3´
F: 5´-TCTCTCACGGTCCCCATAAT-3´; R: 5´-AGCAAGCCTAATGACACCTT-3´
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147
|
BM2
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|
Clostridium botulinum
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Random chromosomal DNA fragment‡
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NA
|
P: 5´- CATCTTCACGGAGGTGAACAAGCCTCCATGTTCGACGGTAACCCAGAAGC-3´
F: 5´-AGATCAGAAGGATGTACCGC-3´; R: 5´-ACTCTTGGACATTGGGGTAA-3´
|
173
|
CB1
|
|
P: 5´-CCACCGGGAGTGCCGGAAGATCAGAAGGATGTACCGCTAGTAACTCATCT-3´
F: 5´-AATAATACGCCTATGACGCC-3´; R: 5´-TAAACCTTTTGCTGTCCACC-3´
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163
|
CB2
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Yersinia pestis
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Plasminogen activator
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M27820
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P: 5´-GGACGTCTCTGGCTTCCGGGTCAGGTAATATGGATGACTACGACTGGATG-3´
F: 5´-CAAAAATGTCGCTATCCTGA-3´; R: 5´-GGTCATATTCATTGGCATGA-3´
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201
|
YP1
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P: 5´-TCCATACTCATTTCTGACCCTGAATGCCAGGGGGTGGACGTCTCTGGCTT-3´
F: 5´-ACGCAGAAACAGGAAGAAAG-3´; R: 5´-CATCCAGTCGTAGTCATCCA-3´
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168
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YP2
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Bacillus thuringiensis kurstaki
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cry1A
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AY225453
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P: 5´-TGGTCAGGGCATCAAATAATGGCTTCTCCTGTCGGTTTTTCGGGGCCAGA-3’
F: 5´-TATTATTGGTCAGGGCATCA-3´; R: 5´-GTTCTATACACGCCCTGACC-3´
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144
|
BTK1
|
|
P: 5´-GGTAGTTTTCGAGGCTCGGCTCAGGGCATAGAAAGAAGTATTAGGAGTCC-3´
F: 5´-TGATGGTAGTTTTCGAGGCT-3´; R: 5´-CGACAGGAGAAGCCATTATT-3´
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153
|
BTK2
|
|
Vaccinia virus
|
Thymidine kinase
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U94848
|
P: 5´-CGTATGGCAAACGAAGGAAAAATAGTTATAGTAGCCGCACTCGATGGGAC-3´
F: 5´-ATTCTGTGAGCGTATGGCAA-3´; R: 5´-TCTATCTCGGTTTCCTCACC-3´
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199
|
VA1
|
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P: 5´-GGCGGACATATTCAGTTGATAATCGGCCCCATGTTTTCAGGTAAAAGTAC-3´
F: 5´-GGCGGACATATTCAGTTGAT-3´; R: 5´-GCTTCCAATGCTTCAAAATT-3´
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183
|
VA2
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Francisella tularensis
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fopA
|
AF097542
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P: 5´-GGTTCAGCTACAGCATCTATCGCTGCAGGTTCAGATAATATCGATACATC-3´
F: 5´-TTAGGTTCAGCTACAGCATC-3´; R: 5´-TGTCCTTGTTAGTCAAAGCG-3´
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155
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FT1
|
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P: 5´-CATCAGCAAACACTAATTCAGCTACTACACAAAGCAGTGGTTTTGCAGCT-3´
F: 5´-TCAGCTACAGCATCTATCGC-3´; R: 5’-TGTCCTTGTTAGTCAAAGCG-3’
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149
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FT2
|
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*P, probe; F, forward primer; R, reverse primer;
NA, not applicable. All sequences were purchased from Integrated
DNA Technologies, Inc. (Coralville, IA, USA). Probe sequences were
modified with an amine group and C6 linker at the 5´ position,
and these were used to immobilize the probe on microspheres. Synthetic
complementary targets of probes and reverse primers were modified
with Cy3 at 5´ position as a reporter for array hybridization.
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†Sequences are from the noncoding region between
2 open reading frames, BMEI1658 and BMEI1659.
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‡Sequences were obtained by sequencing by the collaborator
and are not available from GenBank.
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Appendix Table 2. Cross-reactions
between 18 probes on the multiplexed array* (Download
PDF [93
KB, 1 page])
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Targets (100 nmol/L)
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Probes
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|
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Bacillus anthracis
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Brucella melitensis
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Bacillus thuringiensis kurstaki
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Yersinia pestis
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Vaccinia virus
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Francisella tularensis
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Clostridium botulinum
|
|
|
|
|
|
|
|
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capA
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pagA
|
SASP-B
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Sequence between BMEI1658 and BMEI1659
|
Cry1A
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plasminogen activator
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Thymidine kinase
|
fopA
|
Random chromosomal DNA fragment
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|
|
|
|
|
|
|
|
|
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BA1
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BA2
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BA3
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BA4
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BA5
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BA6
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BM1
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BM2
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BTK1
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BTK2
|
YP1
|
YP2
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VA1
|
VA2
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FT1
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FT2
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CB1
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CB2
|
|
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BA1
|
808.1
|
173.4
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13.8
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14.5
|
18.1
|
9.61
|
17.1
|
9.75
|
21.1
|
21.3
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22.2
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27.9
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31.8
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29.2
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30.8
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27.4
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30.1
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27.2
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BA2
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712.3
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701.2
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15.1
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18.1
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9.57
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–0.28
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11.9
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–1.54
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25.8
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15.6
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19.8
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13.4
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15.2
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32.5
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10.5
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5.2
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21.6
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23.1
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BA3
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9.61
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13.4
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457.4
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6.48
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6.15
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7.6
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12.4
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11.4
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–2.69
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13.9
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5.56
|
6.66
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11.3
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24.1
|
8.61
|
–2.84
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29.8
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16.9
|
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BA4
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11.7
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20.8
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26.9
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693.2
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26.6
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8.61
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20.3
|
25.5
|
31.6
|
14.4
|
5.65
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9.05
|
19.6
|
21.6
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17.9
|
31.1
|
19.8
|
25.6
|
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BA5
|
10.9
|
2.4
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5.7
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14.2
|
410.3
|
113.6
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10.7
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2.2
|
12.6
|
19.8
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25.7
|
30.1
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–1.6
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21.7
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28.2
|
25.8
|
30.1
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8.78
|
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BA6
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17.7
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–3.45
|
11.3
|
15.5
|
262.8
|
853.9
|
17.5
|
31.6
|
29.9
|
11.4
|
33.6
|
32.2
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24.8
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27.6
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30.9
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11.9
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13.7
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31.4
|
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BM1
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7.92
|
3.32
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23.2
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–3.22
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18.8
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11.5
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723.5
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–4.28
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32.2
|
–4.99
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20.8
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25.8
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18.2
|
17.3
|
29.3
|
27.9
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25.9
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14.6
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BM2
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3.08
|
3.84
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26.8
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6.78
|
3.96
|
5.67
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24.1
|
973.3
|
21.3
|
1.11
|
15.9
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17.6
|
19.9
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32.8
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13.9
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24.4
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34.4
|
12.9
|
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BTK1
|
14.1
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6.1
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30.4
|
6.35
|
15.6
|
9.77
|
17.1
|
6.05
|
1423.1
|
232.8
|
32.4
|
19.2
|
19.7
|
12.5
|
16.6
|
26.3
|
8.95
|
23.1
|
|
BTK2
|
27.7
|
–0.91
|
3.39
|
6.8
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8.39
|
4.56
|
10.3
|
9.59
|
425.8
|
1288.6
|
22.1
|
8.16
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9.8
|
18.9
|
28.2
|
19
|
7.84
|
19.4
|
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YP1
|
12.5
|
–0.79
|
28
|
4.56
|
12.5
|
5.11
|
8.88
|
20.6
|
12.9
|
32.1
|
2089
|
588.7
|
23.6
|
24.6
|
179.7
|
25.7
|
15.9
|
29.2
|
|
YP2
|
23.4
|
32.1
|
23.6
|
14.2
|
–1.58
|
29.3
|
15.4
|
19.1
|
28.1
|
23.8
|
1491.5
|
1784
|
14.5
|
11.3
|
9.7
|
7.05
|
23.9
|
14.6
|
|
VA1
|
8.9
|
24.9
|
25.3
|
4.71
|
8.61
|
18.7
|
13.6
|
8.61
|
3.12
|
9.87
|
12.7
|
16.6
|
1536.9
|
1.75
|
25.4
|
23.8
|
17.7
|
11.8
|
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VA2
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43.1
|
15.9
|
4.59
|
8.94
|
7.18
|
23.2
|
29.4
|
3.25
|
15.4
|
14.7
|
26.7
|
12
|
18.3
|
1703.4
|
23.9
|
–0.76
|
26.3
|
29.7
|
|
FT1
|
19.8
|
19.7
|
8.77
|
25.6
|
14.6
|
4.65
|
32.1
|
14.7
|
6.96
|
1.56
|
14.9
|
0.65
|
0.56
|
0.78
|
793
|
26.8
|
28.5
|
20.5
|
|
FT2
|
25.4
|
8.4
|
11.5
|
16.9
|
19.7
|
7.98
|
9.94
|
17.9
|
12.5
|
25.1
|
4.44
|
14.5
|
8.3
|
13.4
|
216.7
|
456.9
|
8.99
|
27.9
|
|
CB1
|
23.1
|
5.68
|
16.9
|
20.1
|
27.2
|
15.3
|
14.1
|
9.11
|
7.46
|
16.1
|
4.6
|
19
|
28.3
|
171.6
|
4.93
|
24.8
|
843.1
|
18.5
|
|
CB2
|
14.8
|
11.2
|
8.45
|
8.16
|
5.45
|
24.6
|
20.9
|
3.25
|
1.62
|
21.9
|
25.8
|
23.5
|
13.3
|
18.0
|
28.2
|
21.2
|
89.1
|
592.1
|
|
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*The numbers are net fluorescence intensities (in
arbitrary units) obtained by subtracting the average intensity value
of the background from that of the hybridization signal. The specific
response for each probe is indicated in red.
The cross responses between probes designed for the same biological
warfare agent (BWA) are indicated in pink,
and the cross-reactions between probes from different BWAs are marked
with blue. The standard deviation (SD)
of background is 15 (n = 3), and the detection limit of array hybridization
for each microsphere probe is 45, defined as 3 × SD.
|
|
Appendix Table 3. Number
of microorganisms in autoclaved bacterial cultures used as templates
in polymerase chain reaction; cultures were also used to spike wastewater
and prepare mixed bacterial samples*
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|
|
Organism
|
DNA copies/mL
|
|
|
Bacillus thuringiensis kurstaki
|
2.4 × 108
|
|
B. anthracis Ames
|
1.2 × 105
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|
Vaccinia virus
|
7.7 × 106
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|
Yersinia pestis CO-92
|
2.8 × 105
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|
Clostridium botulinum A
|
3.1 × 107
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|
Francisella tularensis Novicida
|
1.1 × 106
|
|
|
*Naval Medical Research Command (NMRC) did not provide
an autoclaved bacterial culture of Brucella melitensis; therefore,
no results were obtained for Brucella melitensis other than
with synthetic target sequences. The numbers provided by NMRC in
this table are based on real- time polymerase chain reaction quantification,
where a standard curve was produced with cloned material and used
to calculate the results of a gene copy/mL quantification of the
autoclaved material.
|
|
