title: Rapid and simultaneous screening of 47,XXY and AZF microdeletions by quadruplex real-time polymerase chain reaction.
abstract: We developed a quadruplex real-time PCR assay that allows rapid and simultaneous detection of 47,XXY and azoospermia factor (AZF) microdeletions on Y chromosome. The quadruplex assay consisted of four hydrolysis probes and primer sets. Three probes and the corresponding primers were used to qualitatively detect AZFa, AZFb, and AZFc deletions. For the detection of 47,XXY, the hydrolysis probe-mediated melting analysis was conducted to analyze the relative amounts of X and Y chromosomes. The quadruplex assay for detecting 47,XXY was characterized by very high analytical specificity (100%) in a wide template DNA range (2-100 ng). The detection limit of the assay was 2 ng of genomic DNA, and the optimal template DNA amount for the detection of 47,XXY was 25 ng. The quadruplex assay for detecting 47,XXY and AZF microdeletions has also demonstrated very high diagnostic sensitivity and specificity (100%). The assay was found to be rapid, sensitive, reliable, and inexpensive. This method is suggested to be applied as a first-step tool in genetic screening of patients with non-obstructive azoospermia and severe oligospermia.
authors.affiliation: Molecular Diagnostics Laboratory, Department of Medical Genetics, Prenatal Diagnosis Center of Xiamen, Maternal and Child Health Hospital, Xiamen, Fujian, China.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Molecular Diagnostics Laboratory, Department of Medical Genetics, Prenatal Diagnosis Center of Xiamen, Maternal and Child Health Hospital, Xiamen, Fujian, China.
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Molecular Diagnostics Laboratory, Department of Medical Genetics, Prenatal Diagnosis Center of Xiamen, Maternal and Child Health Hospital, Xiamen, Fujian, China.
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Molecular Diagnostics Laboratory, Department of Medical Genetics, Prenatal Diagnosis Center of Xiamen, Maternal and Child Health Hospital, Xiamen, Fujian, China. Electronic address: guoqiwei@gmail.com.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Guo
mesh_terms: D057205:Automation, Laboratory; D053713:Azoospermia / Q000209:etiology; D002681:China; D002872:Chromosome Deletion; D041322:Chromosomes, Human, Y / Q000235:genetics / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007248:Infertility, Male; D057230:Limit of Detection; D008297:Male; D060885:Multiplex Polymerase Chain Reaction; D009845:Oligospermia / Q000209:etiology; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012729:Sex Chromosome Aberrations; D058533:Sex Chromosome Disorders of Sex Development / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology
keywords: 47,XXY; AZF microdeletion; Azoospermia; Oligospermia; Real-time PCR