Innovative use of platinum compounds to selectively detect live
microorganisms by polymerase chain reaction
Supplementary Material
Takashi Soejima *1, Jun-ichi Minami1, Jin-zhong Xiao1, Fumiaki Abe1
1
Biological Function Research Department, Food Science & Technology Institute, Morinaga
Milk Industry Co., Ltd
* Corresponding author:
Takashi Soejima.
5-1-83 Higashihara, Zama City
Kanagawa Pref., 252-8583, Japan
phone: +81 46 252 3066; fax: +81 46 252 3059;
E-mail address: t_soezim@morinagamilk.co.jp
Running title: Pt compounds to discriminate live from dead bacteria
Data processing with Ct values obtained from live E. coli in milk by Pt-direct-qPCR
using a large milk volume of 12 mL
Live E. coli cells or dead E. coli (boiled for 3 min) were inoculated to one kind of
commercial pasteurized milk (12-mL) at a concentration of 1.0 to 6.4 log10 CFU or cells mL-1.
Following to “Milk pre-treatment” procedure in “Materials and Methods” section in main text,
after exposure of sampled milks to 150 µmolL-1 Pt(PPh3)4(0) or 300 µmolL-1PMA, the
direct-qPCR (50 cycles) was performed in duplicates to obtain raw data of Ct values. The
data processing to obtain the live cell counts for Pt-direct-qPCR, PMA-direct-qPCR, or
direct-qPCR without any Pt and PMA subjections is presented below:
In relation to the assay for Pt-direct-qPCR, the live counts were calculated from the
obtained Ct values by using the regression line of Figure 2B (y = -3.45x + 42.82).
Likewise,
with respect to the assay for PMA-direct-qPCR, the live counts were calculated from the
obtained Ct values by using the regression line of Figure 2C (y = -4.40x + 51.05). Similarly,
concerning the assay for direct-qPCR, the live counts were calculated from the obtained Ct
values by using the regression line of Figure 2B (y = -2.60x + 37.55) obtained at a range from
3 to 6 log10 CFU mL-1 labelled as Live (No-agent). Then, the dead counts were calculated
using the regression line of Figure 2B obtained at the same range labelled as Dead (No-agent).
Additionally, as for the assay data presented in Supplementary Table 2, the milk samples
exogenously added with live E. coli at a concentration of 5.1 to 6.4 log10 CFU mL-1 followed
by the treatment with the Pt compound, PMA agent, or no any Pt and PMA agents were
finally diluted in the sterile water (SW) by 10-fold (that is, 1/10 amounts of finally obtained
pellets for test sample, considering the range of linearity presented in Figure 2B and 2C.
For a typical culture method, directly or appropriately diluted milk samples were cultured by
VRBA medium.
Discriminating between live and dead C. sakazakii by DNA-alkylating agents combined
with the direct-qPCR
The cyclophosphamide monohydrate ISOPAC®, melphalan, and chlorambucil (Sigma, St.
Louis, MO, USA), which are agents used to alkylate and cross-link DNA (Rad et al., 2003;
Russell et al., 1992; Shand et al., 1978), were, respectively dissolved in DMSO at an initial
concentration of 10 mM; afterwards, the solutions were diluted with physiological saline to
concentrations ranging from 100 to 5000 µM.
Hereafter, the addition of their
DNA-alkylating agents to live or dead C. sakazakii suspension with SW, and experimental
procedures such as the exposure condition followed the “Preparation of platinum compounds
and PMA and exposure methods” in the Material and Methods section in main text.
Then,
successive Direct qPCR procedures for pellets obtained after centrifugation at 3,000 × g for 5
min at 4°C followed the “Direct qPCR without the need for DNA extraction of bacteria (live
and dead cells) treated with or without Pt compounds compared with PMA agents” in
Materials and Methods section in main text.
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Supplementary Table captions
Supplementary Table 1
Comparison of Pt-direct-qPCR with other typical methods
without pre-incubating milk samples.
The value of 0.0: no counts (VRBA) or no elongation (Pt-direct-qPCR and
PMA-direct-qPCR) but not 0.0 log10 CFU or cells mL-1 (100 = 1).
The description of 0 ˂ ˂ 3 (+) for qPCR: the cells number was between 0 and 3 log10 CFU or
cells mL-1, but accurate counts were not done owing to rude regression line as presented in
plot data labelled as Live (No-agent) or Dead (No-agent) of Fig. 2B.
Supplementary Table 2
Comparison of Pt-direct-qPCR with other typical methods with
2-h pre-incubation milk samples.
The value of 0.0: no counts (VRBA) or no elongation (Pt-direct-qPCR and
PMA-direct-qPCR) but not 0.0 log10 CFU or cells mL-1 (100 = 1).
The description of 0 ˂ ˂ 3 (+) for qPCR: the cells number was between 0 and 3 log10 CFU or
cells mL-1, but accurate counts were not done owing to rude regression line as presented in
plot data labelled as Live (No-agent) or Dead (No-agent) of Figure 2B.
Supplementary Table 3
Cross-table between each method with and without
pre-incubating milk samples.
Supplementary Table 4
Discrimination between live and dead C. sakazakii
by DNA-alkylating agents followed by the direct-qPCR.
Dead: Dead cells were prepared by boiling for 3 min followed by immediate chilling.
Live (Ct) and Dead (Ct): the direct-qPCR (50 cycles) was performed in duplicate, and the
Ct values are presented as the average ± SD (n = 2).
Supplementary Figure 1.
Pt compounds used in this study.