非梗阻性无精少精症主要遗传因素的快速检测新方法研究

Klinefelter syndrome (KS) and Y-chromosomal microdeletions (YCMD) are major genetic causes of non-obstructive azoospermia or oligospermia in infertile men. Diagnosis of KS and YCMD can in?uence therapeutic options, and has prognostic value. Currently, there are several ways to diagnose these two diseases. However, performance or throughput limitations of these methods constrain their use in routine test..To address this problem, we try to develop a novel method which can simultaneously detect KS and YCMD in a single reaction vessel. In this method, we will set up a quadruplex real-time PCR assay. Triplex reactions in this assay are designed to detect the microdeletions in AZFa, AZFb, and AZFc regions, respectively. At the meantime, KS can be diagnosed by the probe-melting analysis in the same reaction vessel. The total turnaround time of this assay is approximately 1.5 hours. In comparison with conventional methods, our method can reliably diagnose KS and YCMD simultaneously, while markedly simplifying the assays and reducing time and cost. Furthermore, our method will be amenable to automation and provides sufficient capacity for high throughput. Thus, our method will be very suitable for routine detection for non-obstructive azoospermia or oligospermia patients.

非梗阻性无精少精症是导致男性不育的重要原因。克氏综合征与Y染色体微缺失是造成非梗阻性无精少精症的主要遗传因素，对于这两个遗传因素的检测能够有效指导男性不育的科学治疗与预后。目前，针对克氏综合征与Y染色体微缺失的诸多检测方法均具有一定缺陷，无法满足快速、准确、高通量的检测需求。.本项目拟建立一种基于实时荧光PCR平台的检测新技术，能够在一个反应管内快速准确地同时检测克氏综合征与Y染色体微缺失。该技术拟建立一个高效的四重实时PCR反应体系。其中三重反应可分别指示Y染色体上AZFa、AZFb、AZFc三个亚区的缺失情况，另外一重反应可结合探针熔解曲线技术检测克氏综合征的存在情况。整个反应耗时约1.5个小时，无需PCR产物的开放式分析，避免了污染的发生。同时该技术易于自动化，具有极高的检测通量，能够满足大规模样本检测的需求。本项目立足国情，有望成为非梗阻性无精少精症的常规检测技术。

非梗阻性无精少精症是导致男性不育的重要原因。克氏综合征与Y染色体微缺失是造成非梗阻性无精少精症的主要遗传因素，对于这两个遗传因素的检测能够有效指导男性不育的科学治疗与预后。目前，针对克氏综合征与Y染色体微缺失的诸多检测方法均具有一定缺陷，无法满足快速、准确、高通量的检测需求。.本项目建立了一种基于实时荧光PCR平台的检测新技术，能够在一个反应管内快速准确地同时检测克氏综合征与Y染色体微缺失。该技术拟建立一个高效的四重实时PCR反应体系。其中三重反应可分别指示Y染色体上AZFa、AZFb、AZFc三个亚区的缺失情况，另外一重反应可结合探针熔解曲线技术检测克氏综合征的存在情况。整个反应耗时约1.5个小时，无需PCR产物的开放式分析，避免了污染的发生。.实验表明，我们的技术获得了100%的分析特异性，具有很宽的检测范围（2-100 ng DNA），最适检测量为25 ng DNA。在临床实验中，本技术获得了100%的诊断灵敏度和特异性。.该技术易于自动化，具有极高的检测通量，能够满足大规模样本检测的需求，立足国情，有望成为非梗阻性无精少精症的常规检测技术。