Kits and Instrument for Microcystis and Microcystins detection

摘要

Microcystins (MCs), include the Microcystin-LR (MC-LR) and the Microcystin derivatives, are cyanobacterial hepatotoxins responsible for animal-poisoning and human health incidents. So, it is very necessary to develop sensitive analysis methods for MC-LR and MCs-total in surface water as well as drinking water. At the same time, it is also very critical to quantitatively detect the most concerned cyanobacteria -microcystis, include the toxin-producing microcystis and total microcystis. 1) Immunoassays provide a sensitive and fast means to detect these toxins. We have produced and characterized two kinds of monoclonal antibody (Clone MC8C10 and MC10F2) with high affinity to microcystins, MC8C10 is very specific against MC-LR individual, and MC10F2 has Broad-spectrum specificity with all [4-arginine] microcystins. Then, based on the one-step competitive enzyme-linked immunosorbent assay (ELISA), two kinds of ELISA kit were established to specifically detect the MCs-total and MC-LR individual, the quantitative detection range is from 0.1 μg L-1 to 4 μg L-1. 2) Fast and sensitive detection of microcystin-LR (MC-LR) was conducted with a portable microcystins analyzer, which was based on the principle of immunoassay and total internal reflection fluorescence. The reusable fiber optic probe surface was produced by covalently immobilizing a MC-LR-ovalbumin (MC-LR-OVA) conjugate onto a self-assembled thiol-silane monolayer of fiber optic probe through a hetero-bifunctional reagent. The fiber optic probe has high recoveries (more than 150 assay cycles). One assay cycle including surface regeneration was less than 15 min. For the standard curve, the limit of detection (LOD) of 0.03 μg L-1 and the quantitative detection range was 0.1-10 μg L-1. 3) Real-time quantitative PCR (rt-qPCR) was developed to quantify total and microcystin-producing microcystis cells in natural samples using specific primers of 16S rRNA and mcyA genes, respectively. The standard curve using standard plasmid DNA showed that the reliable detection ranging from 1× 102 to 1× 108 copies per qPCR reaction for both 16S rRNA and mcyA genes of microcystis. 4) The two kinds of ELISA kit, the portable and easy-to-use microcystins analyzer, and the two kinds of rt-qPCR kit, can be commercially obtained from the company J&Q Environmental Technologies Co., Ltd. (www.jq-environ.com, Beijing, China).