Serratia marcescens--all strains
Sample Preparation
Because of the potential for PCR inhibitors in host tissue, isolate to improve detection. Dissect
vascular bundles from a lower stem section scrubbed with 70% ethanol, place in a 1.5 ml epitube containing 250 l Nanopure water, crush with a sterile golf tee, vortex, and let sit for 30+
minutes (as long as 24 h). Mix suspension and streak onto LB agar or nutrient agar and incubate
for 48 hours. Suspend bacterial growth in 1-2 ml Nanopure water; if the cell suspension is
completely opaque, dilute until some translucency is achieved. In most instances, a 1/10 dilution
is sufficient; some samples need as much as 1/1000 dilution.
Reaction Mixture (per 25 l tube)
Reaction master mix may be prepared in advance, in darkened conditions, and stored
at -18ºC without loss of sensitivity for up to 4 d (the longest intervals tested thus far).
6 l Nanopure water
5 l Smart Cycler Additive Reagent
2.5 l of 2 mM dNTP=s
2.5 l primer YV1
2.5 l primer YV4
2.5 l 10X buffer
0.5 l Titanium Taq polymerase (=0.5 units)
2.5 l of fresh 5X SYBR Green
Sample
1.0 l of sample (or controls as noted below)
Primers
Prepare to a concentration of 0.8 M (0.8 pmol/l)
YV1 5'-GGG AGC TTG CTC CCC GG-3'
YV4 5'-AAC GTC AAT TGA TGA ACG TAT TAA GT-3'
Controls
Negative control:
Positive control #1:
Positive control #2:
1 l Nanopure water
1 l of 100 pg/l of genomic DNA of S. marcescens 102 or 103.
1 l of 100 pg/l of genomic DNA of S. marcescens 102 or 103 plus 1 l
of sample. If results show evidence of PCR inhibition, retest sample
extract at 10-fold dilution.
Cycling Protocol
Name of Protocol: Serratia marcescens YV
1. 95 C for 180 sec
2. 45 cycles of:
95 C for 30 sec
55 C for 30 sec
72 C for 120 sec
3. Final extension
72 C for 600 sec
4. Melt curve from 60 C to 95 C, ramp rate 0.2 C/sec
Duration of protocol: 2.75 h
Expected Results
Product is 409 bp long, with a Tm of 88.4-90.1ºC. Template-free controls in this assay have had
a Tm of 62.3-64.4ºC. Non-specific products have had Tm values of 75.6-78.6ºC; some assays
have yielded specific products of approximately 50 bp with Tm values of 78.0-81.1ºC. Limit of
sensitivity with purified DNA is 10 pg genomic DNA per tube.
Cells of Serratia marcescens appear to have thermostable nuclease enzymes that degrade the a79
amplicon, so put reaction tubes in ice immediately after PCR has run and gel the gel the same
day, if needed. Otherwise, store PCR amplicons at -20 ºC. This has not been a problem with
PCR runs using extracted DNA as the source of template.
Reference
Benny Bruton, pers. comm., 2003.
Document Control: 2/13/16