|
課題背景：由2010年10月-2011年3月來中國醫(yī)科大學(xué)附屬盛京醫(yī)院輔助生殖門診78例男性不育患者作為研究對象, 采用精液常規(guī)檢測精子情況, 并檢測患者性激素水平。選擇出無精子癥患者（18例）和少精子癥患者（20例），對這些患者進(jìn)行EDTA抗凝靜脈血提取DNA通過QF-PCR(Quantitative fluorescence PCR, QF-PCR)進(jìn)行基因檢測。 研究方法：通過對無精和少精患者進(jìn)行QF-PCR檢測，以無精和少精患者外周血基因組DNA 為模板選擇X,Y染色體雜合度較高的STR(含AZFc 區(qū)STR DYS448)和特異性位點(diǎn)，以及SRY 基因。設(shè)計(jì)引物并用不同顏色的熒光在其正向引物5′末端進(jìn)行標(biāo)記，通過多重PCR擴(kuò)增AZFa、AZFb、AZFc區(qū)各2個序列標(biāo)簽位點(diǎn)(Sequence tagged sites, STS),并以健康已生育男性DNA為陽性對照, 女性DNA為陰性對照。通過PCR產(chǎn)物經(jīng)瓊脂糖電泳檢測熒光強(qiáng)度，來判斷AZF微缺失和染色體突變。 研究結(jié)果：由于AZF區(qū)域是否發(fā)生微缺失與無精子癥密切相關(guān)，可以用QF-PCR方法在AZFa、AZFb、AZFc 3個區(qū)分別設(shè)計(jì)熒光標(biāo)記引物sY84和sY86、sY127和sY134、sY254和sY255來檢測AZF區(qū)域是否存在染色體細(xì)微結(jié)構(gòu)的異常,來進(jìn)行男性不育的遺傳學(xué)診斷。同時通過基因?qū)φ諄韺ふ页鋈旧w突變（47, XXY 患者和46, XX 性反轉(zhuǎn)患者），并且可以區(qū)分出46,XX(SRY+)和46, XX(SRY-)患者。 點(diǎn)評：本文通過用QF-PCR進(jìn)行多位點(diǎn)基因檢測并進(jìn)行分析，更迅速、直接地檢測到染色體異常區(qū)域?？梢员苊馀R床上臨床上由于診斷醫(yī)師經(jīng)驗(yàn)不足造成誤診和延后診斷。但是通過文章可以看出由于QF-PCR還會受到檢測位點(diǎn)選擇、STR位點(diǎn)雜合度等因素的影響，所以對臨床患者選擇方面會存在一定的局限性。天昊公司的拷貝數(shù)變異檢測專利技術(shù)AccuCopy可以替代傳統(tǒng)的定量PCR，事實(shí)上，在男性不孕不育方面，天昊公司已經(jīng)使用Accucopy技術(shù)與上海的科研單位合作了相關(guān)課題，從數(shù)據(jù)來看。AccuCopy技術(shù)具有更為準(zhǔn)確，更高通量，高靈活性，更低成本等優(yōu)勢。 Abstract: To assess the clinical practice of quantitative fluorescence PCR (QF-PCR) in genetic diagnosis of male infertilitypatients, 78 nonobstructive male infertility patients were pooled for semen routine screening and sexual hormone determination;QF-PCR was applied to detect the polymorphic short tandem repeat (STR) and specific sequence tagged site (STS)of sex chromosomes; routine chromosome G-band was used for karyotype analysis and PCR was used for the detection ofAZF microdeletion. Routine screening of semen found 18 azoospermia and 20 oligospermia patients (48.72%). Three patientswith 47, XXY, two with 46,XX(SRY+)and one with AZFc microdeletion were detected using QF-PCR techniquewhich were verified by chromosome G-band and PCR. This study suggests that QF-PCR is a comprehensive, rapid andreliable method for detecting abnormal chromosomal regions and microstructures compared with traditional tests and provides a better candidate for diagnosis of male infertility caused by chromosomal anomalies and gene mutation. |
