Establishment of double probes rolling circle amplification combined with lateral flow dipstick for rapid detection of Chattonella marina

Chattonella marina is one of the main algae that could cause harmful algal blooms. It has killed a large number of cultured fish in coastal areas of many countries, causing serious economic losses. Therefore, it is necessary to establish a method that can specifically detect C. marina at pre-bloom abundance, so that timely measures can be taken before this alga causes harm. In this study, a long probe, a short probe and a pair of amplification primers were first designed by using the internal transcribed spacer (ITS) sequence of C. marina as the target gene and using the CD74 gene of a distant species Gallus gallus as the base sequence. The double probes rolling circle amplification (dpRCA) system was then established with the designed probes and amplification primers. A novel detection protocol referred to as dpRCA-LFD by combining the dpRCA products and lateral flow dipstick (LFD) was finally established, which can make the detection results visible to the naked eyes. The reaction conditions of dpRCA were optimized and the optimal conditions were as follows: cycle number of ligation reaction, 12; ligation temperature, 58 degrees C; amplification temperature, 60 degrees C; and amplification time, 60 min. The specificity test that was performed using the optimized dpRCA conditions indicated that dpRCA-LFD was exclusively specific for the target alga. The tests with the genomic DNA of C. marina and the recombinant plasmid containing the ITS sequence of C. marina showed that the sensitivity of dpRCA-LFD was 100 times higher than that of conventional PCR. The detection limit (DL) for the genomic DNA was 8.3 x 10(-3) ng mu L-1 (8.3 x 10(-3) ng per reaction), and the DL for the recombinant plasmid DNA was 7.8 copies mu L-1 (7.8 copies per reaction). The practicality of the developed dpRCA-LFD was further validated by test with the spiked samples containing C. marina and field samples. The simulative test showed that the dpRCA-LFD has a DL of 10 cells mL(-1). The dpRCA-LFD could successfully recognize the target cells from the field samples. In summary, the dpRCA-LFD established in this study has advantages of good specificity, high sensitivity, and easily visible detection results, and therefore is promising for the analysis of C. marina in field samples.