Detection ofProrocentrum minimumby hyperbranched rolling circle amplification coupled with lateral flow dipstick

A novel method referred to as hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD) (HRCA-LFD) here was developed for specific, sensitive, rapid, and simple detection ofProrocentrum minimum. HRCA-LFD relies on a padlock probe (PLP) consisting of a common ligation sequence, two terminal sequences that complement the target DNA, and a manually designed detection probe (LFD probe). The two terminal sequences of the PLP were designed against the species-specific sites of the large subunit ribosomal DNA (LSU rDNA) D1-D2 region ofP. minimum. The optimum parameters for HRCA were as follows: PLP concentration of 20 pM, ligation time of 30 min, ligation temperature of 59 degrees C, enzymic digestion time of 105 min, amplification time of 45 min, and amplification temperature of 58 degrees C. The HRCA-LFD displaying high specificity could accurately distinguishP. minimumfrom other microalgae. The detection limit of HRCA-LFD was as low as 1.42 x 10(-7) ng mu L(-1)for genomic DNA, 1.03 x 10(-7) ng mu L-1(approximately 27 copies) for recombinant plasmid containing the inserted LSU rDNA D1-D2, and 0.17 cells for crude DNA extract ofP. minimum, which was consistently 100 times more sensitive than regular PCR. Interfering test suggested that the performance of HRCA-LFD is stable and would not be affected by other non-target species. The HRCA-LFD results of field samples that are comparable with microscopic examination confirmed that the developed method is competent for detection of target cells in field samples. In conclusion, the developed HRCA-LFD exhibiting stable performance is specific, sensitive, and rapid, which provides a good alternative to traditional microscopic examination for the detection ofP. minimumcells in field samples.