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Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay

First and second authors: Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Science, Beijing 100080, P.R. China; first and fourth authors: Department of Plant Pathology, Shanxi Agricultural University, Taigu, Shanxi 030801, P.R. China; and third author: Beijing Animal and Plant Quarantine Bureau, Beijing 100029, P.R. China

The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg²⁺ concentration, and extension temperature were 400 nM, 3.0 mM, and 60°C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.

Additional keywords: cycle threshold , normalized fluorescence .