Easy detection of Prorocentrum donghaiense by polymerase chain reaction-nucleic acid chromatography strip.

Abstract

In recent years, Prorocentrum donghaiense, as a dominant species, has ranked first in terms of cumulative number and area of algal blooms in the East China Sea. In this study, the D1-D2 region of the large ribosomal subunit of P. donghaiense was used as the target gene, and specific primers DH-FP/DH-RP were designed according to the species-specific region of the target gene. An easy, sensitive and visual detection method refered to as polymerase chain reaction-nucleic acid chromatography strip (PCR-NACS) was established for P. donghaiense. The optimized parameters of the PCR amplification system are as follows: primer concentration, 0.15μM; annealing temperature, 62°C; and Mg2+ concentration, 1.5mM. The specificity test showed that PCR-NACS was exlusively specific for the detection of the target algae. The sensitivity test show that the lowest detection limit (LDL) of PCR-NACS was 2.7 × 10-2ng·μL-1 for genomic DNA and 3.58 × 102 copies·μL-1 for plasmid DNA, respectively. The tests using both genomic DNA and plasmid DNA as templates showed that the sensitivity of PCR-NACS was 10 times higher than that of ordinary PCR. The stability test showed that the interfering algal species did not affect the detection of the target algae by PCR-NACS. In addition, the test with simulated natural samples containing target algae showed that the LDL of PCR-NACS could reach 1.27 × 101 cells·mL-1. In summary, the PCR-NACS established in this study may provide a new method for easy identification of P. donghaiense in natural water samples.