Multiple hybridizations using quantum dot-DNA probes for rapid analysis of microbial community on reverse osmosis membrane

Special emphasis has recently laid on the biofouling on RO membrane surface for desalination process to provide a better insight into the microbial community composing of biofilms. However, it appears that conventional RFLP tool, which is used to quantify and identify bacterial population, is inefficient for this study purpose, as it requires intensive time and labor. To meet the requirement of on-site application and a shorter analysis time, detection of microbial community by multiple hybridization using fluorescent Quantum dot (QD)–DNA probes are suggested since the excitation of multi-QD probes is possible with a single excitation light source. In our study, streptavidin-coated QDs 525, 585 and 655 were used to detect three different target bacteria in a single experiment, and each QD streptavidin was conjugated to specific oligonucleotides DNA of each bacteria. The hybridization between mixed target DNA of Mycobacterium sp., Rhodobacter sp. and Bacillus sp. and each complementary QD–DNA probe occurred in a separation-dependent solution phase. In comparison with the conventional RFLP method, it appeared that the application of QD–DNA probe was quick and promising, as it provides the information of mixed bacteria in a single test.

To meet the requirement of on-site application and a shorter analysis time, detection of microbial community by multiple hybridization using fluorescent QD–DNA probes are suggested. Since various sizes of QD with different fluorescence emission colors can be excited at a single wavelength, there is an advantage to significantly decrease the total reaction time. Conventional RFLP tool is inefficient for this study purpose, as the protocol includes number of time-consuming and labor intensive steps. Based on our experience, it took approximately 1 h for template DNA extraction from bacterial samples, 2 h for amplification of 16S rRNA gene-targeted DNA fragments, 1 h for purification of amplified target DNA, 2 h for restriction enzyme treatment and 1 for identification of DNA fragments digested by restriction enzyme. However, it is believed that detection and identification of mixed bacteria using multiplexed hybridization with various QD–DNA probes can be performed within 4 h under thoroughly defined condition. As a result of applying QD 655, QD 588 and QD 525–DNA probes, simultaneous detection for Mycobacterium sp., Rhodobacter sp. and Bacillus sp. was achieved with three separate fluorescence peaks appeared in a single experimental run. Problem remains in the separation of unbound probes from bound ones and that will cause positive errors in the identification of specific bacterial species.