Rapid detection of pathogenic bacteria in surface Rapid detection of pathogenic bacteria in surface water by bacteria universal primerwater by bacteria universal primer

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.

IntroductionIntroduction

Results & DiscussionResults & Discussion

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.

ConclusionsConclusions

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 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 10-fold 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.

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.

Fig 1. PCR amplification results of the four reference strainsM: DNA marker, 1: Shigella dysenteriae, 2: Vibrio cholerae, 3: Salmonella typhrmurium, 4: Escherichia coli., 5: Negative control

PCR amplification and analysis of reference strainsPCR amplification and analysis of reference strains

Determination of detection sensitivity of the Determination of detection sensitivity of the universal primer PCR protocoluniversal primer PCR protocol

PCR analyses and bacteria enumeration of surface PCR analyses and bacteria enumeration of surface water sampleswater 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.

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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).

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Fig. 4 Enumeration of total bacteria and faecal coliform in surface water samples.

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.

Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50478048)