Transcript IWA Poster Template

Rapid detection of pathogenic bacteria in surface
water by bacteria universal primer
Introduction
The increase of urban population often results in higher percentage of untreated sewage into surface waters, and this is probably responsible for the great
number of human morbidities and mortalities worldwide. In particular, waterborne infections such as typhoid fever, cholera, dysentery and traveler’s diarrhea
are caused by different types of bacterial pathogens and thus pose a major public health hazard, especially in developing countries. Regular monitoring of
waterborne pathogens is extremely important for the protection of public health.The present study is a preliminary work to establish a rapid detection method
using PCR with universal primers. The target bacteria are the common pathogenic bacteria in surface water which are related to the most frequently occurred
waterborne deceases.
Results & Discussion
PCR amplification and analysis of reference strains
The universal primer pair was tested by PCR on DNA templates prepared
from Shigella dysenteriae, Vibrio cholerae, Salmonella typhrmurium, and
Escherichia coli. As shown in Fig. 1, a limpid specific strap could be seen
at 320bp for all the four strains on the electrophoregrams. Analysis results
verified that the universal primer pair showed specificities only for their
corresponding target genes.
Fig 1. PCR amplification results of the four reference strains
M: DNA marker, 1: Shigella dysenteriae, 2: Vibrio cholerae, 3: Salmonella
typhrmurium, 4: Escherichia coli., 5: Negative control
PCR analyses and bacteria enumeration of surface
water samples
Using the universal primer PCR protocol mentioned above, PCR analyses
were conducted for water samples collected from the 5 location. For
comparison, bacteria counting was also conducted regarding total bacteria
and faecal coliform. For PCR analyses, bacterial cells were harvested
from the each of the water samples by centrifugation and then total DNA
was recovered by phenol–chloroform extraction. As shown in Fig.3,
among the 5 water samples, PCR products were detected from 4 samples
and their band intensities were in a sequence of No.4 > No.3 > No.1 >
No.5. Regarding sample No. 2 which was collected from Heihe River, a
well protected water source for potable water supply, there were no any
detectable PCR products. Fig. 4 shows the bacteria enumeration results.
The numbers of total bacteria in the 5 water samples were in a sequence
of No.4 > No.1 > No.5 > No.3 > No.1. Regarding faecal coliform, it was
detected only from 2 water samples, namely No.4 and No.3.
Determination of detection sensitivity of the
universal primer PCR protocol
Total bacterium (cfu/mL) mL)
mL)
Fig 3. PCR detection results of surface water sample.ck–: negative control,
1: Chanhe River, 2: Heihe River, 3: Xingqinghu Lake, 4: Secondary effluent
(undisinfected), 5: Beihu Lake, ck+: positive control,(E. coli. 100cfu/100mL).
Faecal coliform (cfu/100mL)
The detection sensitivity of the universal primer PCR for the Escherichia
coli. was examined in serial 10-fold dilutions in autoclaved distilled water.
Cell suspensions containing 2.75×100 cfu/100mL–2.75×106 cfu/100mL
of Escherichia coli. reference strain was harvested by centrifugation. The
total DNA from bacteria at each serial dilution was extracted by phenol–
chloroform and tested with the universal primer PCR. As shown in Fig. 2,
the detection limit of E. coli. was evaluated to be 27.5 cfu/100mL (Fig. 2a,
line 6), and the detection limit of the chromosome DNA for E. coli. was
evaluated to be 250 ng/L (Figure 2b, line 5). Similar results were obtained
in at least four independent experiments.
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Fig. 4 Enumeration of total bacteria and faecal coliform in surface
water samples.
(b)
Fig 2. The PCR sensibility detection of the universal primer.
(a) PCR amplification results of the E.coli suspension under serially 10-fold
diluted in sterile distilled water. M: Marker, 1: 2.75×106 cfu/100mL,
2: 2.75×105 cfu/100mL, 3: 2.75×104 cfu/100mL, 4: 2.75×103
cfu/100mL, 5: 2.75×102 cfu/100mL, 6: 2.75×101 cfu/100mL, 7:
2.75×100 cfu/100mL, 8: Negative control.
(b) PCR amplification results of the total DNA of E.coli. under serially 10fold diluted in sterile distilled water. M: Marker, 1: 2.5mg/L, 2: 250μg/L,
3: 25μ g/L, 4: 2.5μg/L, 5: 250ng/L, 6: 25ng/L, 7: Negative control.
Sample No.4 is the undisinfected secondary effluent from a domestic
wastewater treatment plant. As anticipated, the PCR band intensity of this
sample was very strong and the numbers of total bacteria and faecal
coliform were both very high (about 20000 cfu/mL and 70 cfu/100mL,
respectively). Sample No.3 from which the PCR band intensity was strong
and higher number of faecal coliform was detected (about 55 cfu/100mL)
is the water from the Xingqing Lake where water pollution used to be very
serious before due to intrusion of urban drainage and domestic wastes
and water quality improvement has just been implemented by sediment
dredging and pollution source control. The result indicated that the water
quality is still bacteriologicaly unfavorable. Regarding sample No.1 which
is from a river receiving urban drainage and certain industrial wastewater,
although no faecal coliform was detected, the total bacteria count was as
high as 16000 cfu/mL and PCR products were also detected.
Conclusions
With its rapidness and sensibility for the detection of pathogenic bacteria from surface water, the universal primer PCR method established by this study can
provide a more reliable approach for bacteriological analysis of water environment from the viewpoint of safeguard of public health.
Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50478048)