A real-time qPCR assay to quantify Ophiocordyceps sinensis biomass in Thitarodes larvae

Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the C T values (R (2)=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came body-wall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these results show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.