Rapid and simultaneous screening of 47,XXY and AZF microdeletions by quadruplex real-time polymerase chain reaction

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.