Amplification of single bulb mites by nested PCR: Species-specific primers to detect Rhizoglyphus robini and R. setosus (Acari: Acaridae)

A major problem of using molecular amplicons for DNA amplification in mite systematics is that sufficient template cannot always be acquired from an individual mite. To solve this problem, we developed a nested PCR for DNA amplification of single Rhizoglyphus robini and R. setosus bulb mites. A dilution up to 105 of the DNA from a single egg, larva, nymph or adult contained enough templates for amplification of the target ribosomal region. However, the use of specific primers in the second PCR is necessary to reduce the generation of non-target DNAs from symbiotic organisms. Identification of bulb mites collected from seven sampling locations in Taiwan or of bulb mites that were used in simulated experiments in the presence of host plant tissues was unambiguous with specific PCR primers.