Qingyun Peng

rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia

Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10(-14)g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.

Detection of Ophiocordyceps sinensis in the roots of plants in alpine meadows by nested-touchdown polymerase chain reaction

Ophiocordyceps sinensis, one of the most important income sources of rural Tibetan families, is an entomopathogenic fungus that parasitizes the ghost moth Thitarodes larvae, which live in alpine meadows on the Tibetan Plateau and in the Himalayas. The annual yield of O. sinensis has gradually declined in recent years. However, there is no effective method to sustain or increase the yield of O. sinensis artificially because the life cycle of the O. sinensis anamorph remains unclear. Here we detected O. sinensis in alpine plant roots by nested-touchdown polymerase chain reaction (PCR). Forty-two alpine plant species were screened. The roots from 23 alpine plant species (54.76%) tested positive including 13 families and 18 genera. The detection results indicate that O. sinensis is present in the plant roots during the anamorph life cycle, to deal with harsh conditions in alpine habitats and have an increased opportunity to infect the larvae. The finding provides new information regarding the biology and ecology of O. sinensis that may be used to sustain this valuable resource.

Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR

Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P < 0.05). Our protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

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 CT values (R2=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.