Draft, Do Not Distribute
11 Approved
Variola
SMPR v7.5
Annex V: Bioinformatics Analyses of Signature Sequences Underlying
Variola virus
286
Assays
287
288
In silico
screening will be performed on signature sequences (e.g., oligo primers/probes) to
289
predict specificity to
Variola virus
and inclusivity across all sequenced
Variola virus
strains.
290
291
In silico
results are suggestive of potential performance issues, so will guide necessary additions
292
to the wet screening panels.
In silico
identification of potential cross-reactions (false positives)
293
or non-verifications (false negatives) would identify the relevant strains to be included in the
294
exclusivity or inclusivity panels, respectively, if available.
295
296
A method developer-selected tool to carry out the bioinformatics evaluation should be able to
297
predict hybridization events between signature components and a sequence in a database
298
including available genomic sequence data, using public Genbank nucleotide
299
[
]. The selected tool should be able to identify predicted
300
hybridization events based on platform annealing temperatures, thus ensuring an accurate
301
degree of allowed mismatch is incorporated in predictions. The program should detect possible
302
amplicons from any selected database of sequence.
303
304
Potential tools for
in silico
screening of real-time PCR signatures include:
305
306
·
Simulate_PCR:
307
o
This program will find all possible amplicons and real time fluorescing
308
events from any selected database of sequence.
309
310
·
NCBI Tools:
311
312
·
FastPCR:
313
314
The method developer submission should include:
315
316
·
Description of sequence databases used in the
in silico
analysis
317
·
Description of tool used for bioinformatics evaluation
318
o
Data demonstrating the selected tool successfully predicts specificity that has
319
been confirmed by wet-lab testing on designated isolates
320
§
This data can be generated retrospectively using published assays
321
·
List of additional strains to be added to the inclusivity or exclusivity panels based on the
322
bioinformatics evaluation
323
