Bacteria adsorption on hydrophilic surfaces for the sensitive detection of pathogenic bacteria using a single tube chamber system.
Here, we developed a simple and effective bacterial isolation method that can be directly used for the detection of pathogenic bacteria. This approach only requires a single plastic tube chamber that can performing serial processes such as cell gathering, cell lysis, nucleic acid amplification, and signaling without the need to transfer samples from one chamber to another. A TEOS (tetraethoxysilane) surface was selected for this application because of its superior performance in the amplification process as well as the ability of bacteria to adsorb to its surface, which is necessary for all processes to be performed in single chamber. The optimal aquatic buffer conditions for bacteria adsorption on the hydrophilic surface were determined to be 1% polyethylene glycerol (PEG) and 10 mM MgCl(2) in 100 mM phosphate at pH 4 for the gram negative bacteria, Escherichia coli O157:H7 (E. coli O157:H7) and 10 mM Na(2)SO(4) in 100 mM phosphate in 100 mM phosphate at pH 4 for the gram positive bacteria, Bacillus cereus (B. cereus). When these divalent cation and anion (MgSO(4)) containing acidic solutions were used, 40% of both bacteria adsorbed onto the hydrophilic surface at a loading rate of 2 mL/min after introduction of low concentrations of bacteria. This method was directly employed to detect E. coli O157:H7 in beef using a single plastic tube chamber that was partially filled with nickel micro beads coated with TEOS. In this system, E. coli O157:H7 were lysed by induction heating of the nickel micro beads. The extracted mRNA was readily amplified and detected by adding an isothermal amplification mixture (NASBA, nucleic acid sequence based amplification) containing a hair-loop type reporting probe with FAM and DABCYL. As a result, this highly sensitive sensing tool could detect very low concentrations of E. coli [10(0) CFU/1 g of beef].
Won
Ji Yeong
JY
Department of Chemical and Biomolecular Engineering, Sogang University, #1 Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea.
Min
Junhong
J
Park
Jung-Hwan
JH
D000327:Adsorption; D000818:Animals; D001409:Bacillus cereus / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D058491:Bacterial Load; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods / Q000706:statistics & numerical data; D002417:Cattle; D018914:Cells, Immobilized; D020099:Coated Materials, Biocompatible; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification; D005516:Food Microbiology; D057927:Hydrophobic and Hydrophilic Interactions; D008460:Meat / Q000382:microbiology; D009532:Nickel; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012821:Silanes
D020099:Coated Materials, Biocompatible; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012821:Silanes; C040733:tetraethoxysilane; D009532:Nickel