Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast.

Harmful algal blooms (HABs) have adverse effects on the marine ecological environment, public health, and marine economy. Thus, methods for the accurate and rapid identification of harmful algal species are urgently needed for the effective monitoring of the occurrence of HABS. A method for the parallel detection of harmful algal species must be established because various HAB-forming algal species coexist in the marine environment. This work developed a multiplex PCR (mPCR) method that can simultaneously detect six common HAB-forming microalgal species distributed along the coast of China: Karlodinium veneficum (Kv), Chattonella marina (Cm), Skeletonema spp., Scrippsiella trochoidea (St), Karenia mikimotoi (Km), and Prorocentrum donghaiense (Pd). Specific mPCR primers were designed from the internal transcribed spacer rDNA or large subunit rDNA gene of the target algal species. The mPCR conditions were optimized. Each mPCR primer was subjected to a cross-reactivity test with other microalgae to confirm the specificity of the developed mPCR system. The results of the system stability test indicated that the background concentration of DNA tested did not affect the performance of the established mPCR system. The results of the sensitivity test showed that the detection limit of the proposed mPCR system for Kv, Cm, Km, and Pd was 0.6 ng µL-1 and that for Skeletonema spp. and St was 0.06 ng µL-1. Additional mPCR analysis with spiked field samples revealed that the detection limit of the mPCR system for Km, Pd, and Kv was 60 cells, whereas that for Cm, Skeletonema spp., and St was 6 cells. The convenience and accuracy of the established mPCR assay were further validated through tests with field samples. The proposed mPCR assay is characterized by parallel analysis, strong specificity, and stability and can be used to supplement morphology-based detection methods for algal species.

[Expressions of LEAFY homologous genes in different organs and stages of Ginkgo biloba].

Expressions of Ginlfy and GinNdly gene were studied by northern blotting in different organs and stages of Ginkgo Biloba. Ginlfy gene was expressed in different organs such as root, stem, leaf of juvenile tree, male tree and female tree, and in different stages of male flower bud and female flower bud. It was inferred that Ginlfy gene could be expressed constitutionally. GinNdly gene was only expressed in leaf of juvenile tree, male tree and female tree and in different stages of male flower bud and female flower bud, while GinNdly gene was not expressed in the other organs. Therefore it was thought that GinNdly gene could be expressed differentially and be a close relation to development of flower.