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Evaluation of immunomagnetic separation for the detection of Salmonella in surface waters by polymerase chain reaction.

Hsu CY, Hsu BM, Chang TY, Hsu TK, Shen SM, Chiu YC, Wang HJ, Ji WT, Fan CW, Chen JL - Int J Environ Res Public Health (2014)

Bottom Line: Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments.These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters.Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

View Article: PubMed Central - PubMed

Affiliation: Division of Urology, Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung 435, Taiwan. jowyu@msn.com.

ABSTRACT
Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR) allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS) theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS) for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

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Sampling points along Puzih River. The sampling sites in the figure are shown as black dots (https://maps.google.com.tw).
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ijerph-11-09811-f001: Sampling points along Puzih River. The sampling sites in the figure are shown as black dots (https://maps.google.com.tw).

Mentions: The Puzih River is 75.67 km long, spanning 426.6 km2 in Chiayi County, mid-southern Taiwan and is an important water source for activities such as agriculture around the area. About two-thirds of the stream was declared to be polluted. Flowing through distinct geographical environments including mountainous countryside, a highly populated city, industrial zones, and costal fish farms, Puzih River has miscellaneous water pollutants ranging from household wastes (59.77%), industry (28.50%), herding or animal husbandry (7.75%), to junkyard leakage (3.98%) [21]. A total of 34 water samples were collected in March 2010 from the Puzih River (23˚28ꞌN, 120˚13ꞌE) in southern Taiwan (Figure 1). For each sample, approximately 2000 mL of water were collected in two sterile 1 L bottles, stored at 4 °C, and transported to the laboratory in 24 h. For concentration of microbes, a one liter sample water was filtered by vacuum through 45-mm diameter GN-6 membranes with a pore size of 0.22 µm (Pall, Mexico City, Mexico) in a stainless steel filter holder. The microbes captured on the surface of the membrane were then eluted by shaking and twisting membranes in 100 mL of sterile phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.5) for 5 min for each sample. The above suspension was transferred into two conical centrifuge tubes (50 mL each) and centrifuged at 5800 g for 30 min at room temperature. After centrifugation, the top supernatant fluid (about 47.5 mL) was aspirated and discarded. The pellet in the remaining 2.5 mL solution was resuspended by vortexing. For each water sample, two tubes of 2.5 mL concentrate were produced for further analyses. For the sake of comparison, a portion of the eluate was collected for IMS processing, and the other was used for Salmonella detection without IMS (Figure 2).


Evaluation of immunomagnetic separation for the detection of Salmonella in surface waters by polymerase chain reaction.

Hsu CY, Hsu BM, Chang TY, Hsu TK, Shen SM, Chiu YC, Wang HJ, Ji WT, Fan CW, Chen JL - Int J Environ Res Public Health (2014)

Sampling points along Puzih River. The sampling sites in the figure are shown as black dots (https://maps.google.com.tw).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199051&req=5

ijerph-11-09811-f001: Sampling points along Puzih River. The sampling sites in the figure are shown as black dots (https://maps.google.com.tw).
Mentions: The Puzih River is 75.67 km long, spanning 426.6 km2 in Chiayi County, mid-southern Taiwan and is an important water source for activities such as agriculture around the area. About two-thirds of the stream was declared to be polluted. Flowing through distinct geographical environments including mountainous countryside, a highly populated city, industrial zones, and costal fish farms, Puzih River has miscellaneous water pollutants ranging from household wastes (59.77%), industry (28.50%), herding or animal husbandry (7.75%), to junkyard leakage (3.98%) [21]. A total of 34 water samples were collected in March 2010 from the Puzih River (23˚28ꞌN, 120˚13ꞌE) in southern Taiwan (Figure 1). For each sample, approximately 2000 mL of water were collected in two sterile 1 L bottles, stored at 4 °C, and transported to the laboratory in 24 h. For concentration of microbes, a one liter sample water was filtered by vacuum through 45-mm diameter GN-6 membranes with a pore size of 0.22 µm (Pall, Mexico City, Mexico) in a stainless steel filter holder. The microbes captured on the surface of the membrane were then eluted by shaking and twisting membranes in 100 mL of sterile phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.5) for 5 min for each sample. The above suspension was transferred into two conical centrifuge tubes (50 mL each) and centrifuged at 5800 g for 30 min at room temperature. After centrifugation, the top supernatant fluid (about 47.5 mL) was aspirated and discarded. The pellet in the remaining 2.5 mL solution was resuspended by vortexing. For each water sample, two tubes of 2.5 mL concentrate were produced for further analyses. For the sake of comparison, a portion of the eluate was collected for IMS processing, and the other was used for Salmonella detection without IMS (Figure 2).

Bottom Line: Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments.These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters.Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

View Article: PubMed Central - PubMed

Affiliation: Division of Urology, Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung 435, Taiwan. jowyu@msn.com.

ABSTRACT
Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR) allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS) theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS) for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection.

Show MeSH