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Can J Microbiol. 2013 Mar;59(3):204-9. doi: 10.1139/cjm-2012-0490. Epub 2013 Jan 18.

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

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1
Food and Health Engineering Research Center of State Education Ministry, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.

Abstract

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.

PMID:
23540339
DOI:
10.1139/cjm-2012-0490
[Indexed for MEDLINE]
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