title: Real-time PCR for sexing Schistosoma mansoni cercariae.
abstract: The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses.
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78245, USA. Electronic address: fcheval@txbiomed.org.
authors.forename: Frédéric D
authors.initials: FD
authors.lastname: Chevalier
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78245, USA. Electronic address: winkal@txbiomed.org.
authors.forename: Winka
authors.initials: W
authors.lastname: Le Clec'h
authors.affiliation: Departments of Biochemistry and Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA. Electronic address: anademattos@cpqrr.fiocruz.br.
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Alves de Mattos
authors.affiliation: Departments of Biochemistry and Pathology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA. Electronic address: loverde@uthscsa.edu.
authors.forename: Philip T
authors.initials: PT
authors.lastname: LoVerde
authors.affiliation: Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX 78245, USA. Electronic address: tanderso@txbiomed.org.
authors.forename: Timothy J C
authors.initials: TJ
authors.lastname: Anderson
mesh_terms: D000818:Animals; D005260:Female; D007814:Larva / Q000502:physiology; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012550:Schistosoma mansoni / Q000254:growth & development* / Q000502:physiology; D012730:Sex Chromosomes; D012732:Sex Determination Analysis
keywords: Cercariae; Schistosome; Sexing methods; qPCR