Non-manual lysis of second-stage Meloidogyne juveniles for identification of pure and mixed samples based on the polymerase chain reaction

Abstract

Non-manual methods of lysing single second-stage juveniles (J2s) of Meloidogyne and direct squashing of nematodes were assessed for consistency by the success of subsequent amplification of mitochondrial DNA by polymerase chain reaction. Microwave heating and boiling resulted in amplification of DNA from only 10% of J2s; treatment with proteinase K, 20%; direct squashing, 50%; and 24 h incubation in NaOH, 81%. Components of mixtures of mtDNA types could be detected consistently by DNA amplification only if they constituted at least 30% of the mixture.