Basic HTML Version
Molecular Pathogens
MP2011, Vol.2, No.3
http://mp.sophiapublisher.com
- 21 -
1.2.2 Sensitivity of
X. axonopodis
pv.
citri
detection
Antiserum 1 and antiserum 2 at the dilution of 1:2,000
were tested for the sensitivity with live and dead cells
of BP210 at different concentrations (10
3
~10
8
CFU/mL).
The positive result is the number which is at least
double the reaction intensity of negative control
(10
6
CFU/mL of
E. coli
and pre-immune serum
reaction). The bacterial suspension of live cells at
10
6
CFU/mL and dead cells at 10
5
CFU/mL showed
positive reaction in ELISA (Table 2). Stronger
reaction can be observed with the increase of bacterial
densities in both antisera tested. Both antisera have the
sensitivity of detection at 10
6
CFU/mL for live cell
and 10
5
CFU/mL for dead cells. The sensitivity of
ELISA for
X. axonopodis
pv.
citri
(Jin et al. 2001),
Clavibacter michiganensis
subsp.
michiganensis
(de
Leon et al., 2008),
C. michiganensis
subsp.
Sepedoni-
cus
and
Erwinia amylovora
(Kokoskova and Mraz,
2008) have also been reported to be at the level of
10
5
~10
6
CFU/mL. The detection efficiency of ELISA
is limited by the level of the pathogen population and
dependent upon the immunological properties of the
antiserum used. The antisera produced in this study
have the sensitivity as high as those produced in other
reports.
Table 2 Sensitivity of ELISA in detecting live and dead cells of
Xac
BP210
Xac
BP210 antigen (CFU/mL)
Antiserum
Cell status
10
8
10
7
10
6
10
5
10
4
10
3
E. coli
10
6
Antiserum 1 (1:2,000)
Live
1.30
0.69
0.23
0.15
0.10
0.10
0.09
Dead
1.99
1.63
0.74
0.21
0.12
0.10
0.09
Antiserum 2 (1:2,000)
Live
0.54
0.47
0.18
0.12
0.10
0.10
0.09
Dead
0.80
0.76
0.63
0.18
0.10
0.10
0.09
Note: The A
405
is shown; 100 µL of the cells were used in each well
1.2.3 Cross-reaction of antiserum to other
Xantho-
monas
sp.
Antisera dilutions (1:1,000 and 1:2,000) were tested
for the cross-reactivity against live cells of other five
Xanthomonas sp.
and
E. coli
at 10
6
CFU/mL. The
results in Table 3 showed that, dilution of 1:1,000
antisera strongly cross-react against
X. axonopodis
pv.
vesicatoria
and weakly reacted against
X. axonopodis
pv.
phaseoli
and
X. campestris
pv.
campestris
. While,
more diluted antisera (1:2,000) cross-reacted with
only
X. axonopodis
pv.
vesicatoria
but not with others
Xanthomonas
(Table 3). These results suggested that,
antisera diluted to 1:2,000 are the suitable condition
for detection of lived target bacteria of at least
Table 3 Sensitivity of ELISA in detecting live and dead cells of
Xac
BP210
Bacterial strain tested
(10
6
CFU/mL)
Diluted antiserum
Antiserum 1
Antiserum 2
(1:1,000) (%)
(1:2,000) (%)
(1:1,000) (%)
(1:2,000) (%)
X. axonopodis
pv.
citri
(BP210)
(Positive control)
100
a
100
100
100
X. axonopodis
pv.
vesicatoria
100
91
80
85
X. axonopodis
pv.
phaseoli
33
0
39
0
X. campestris
pv.
campestris
47
0
27
0
X. axonopodis
pv.
glycine
0
0
0
0
X. oryzae
pv.
oryzae
0
0
0
0
E. coli
(Negative control)
0
0
0
0
Note: a: {[A
405
of each
Xanthomonas
species
-
A
405
of negative control] / [A
405
of positive control
-
A
405
negative control]}
×
100