基于高分辨率熔解曲线检测MD肿瘤微卫星不稳定性  

Detection of Microsatellite Instability in Marek′s Disease Tumor based on High-resolution Melting

作　　者：史泽风 李翎旭 郭译文 廖云 孙昭宇 王来荣 杨德吉[1] 姚大伟[1] SHI Zefeng;LI Lingxu;GUO Yiwen;LIAO Yun;SUN Zhaoyu;WANG Lairong;YANG Deji;YAO Dawei(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2024年第2期759-769,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省自然科学基金(BK20130686)。

摘　　要：本研究旨在通过高分辨率熔解曲线(high-resolution melting,HRM)方法检测马立克病(Marek′s disease,MD)肿瘤中存在的微卫星不稳定现象(microsatellite instability,MSI)。通过临床症状、病理剖检、组织病理学、PCR及测序确定为马立克病。采集5只发病鸡的肌肉、肝、脾、血液及肿瘤组织,提取DNA,采用15对扩增微卫星DNA序列的引物进行HRM检测,得到归一化处理的差异熔解曲线,比较分析各组织熔解曲线的差异,分析15个微卫星标记是否存在基因突变(即MSI)。结果表明,发病鸡的各种临床、病理表现符合为马立克病的特征,病毒meq基因序列与近年来国内流行的MDV强毒株有较高的同源性。HRM分析结果显示,MD发病鸡的肌肉、肝、脾、血液、肿瘤组织中微卫星标记PCR扩增后的熔解曲线存在明显的差异,即存在MSI现象,而且不同的病鸡个体和不同的微卫星标记发生MSI的频率不同。15个微卫星标记在5只鸡的病料中存在MSI的微卫星标记的个数分别为14、7、5、3和1。在15个微卫星标记中,MCWO200、MCW0220这两个微卫星标记在所有的病鸡中都存在MSI,而ADL0158微卫星标记在所有的病鸡中都不存在MSI。通过非变性聚丙烯酰胺凝胶电泳和测序分析进一步证明这些微卫星PCR扩增片段确实存在片段大小不同,碱基序列中存在微卫星重复单位的增多、减少或基因片段的插入或缺失。结果提示,通过HRM分析可以检测MD肿瘤中的MSI现象,是一种灵敏、准确、简便、高通量的方法。The study aimed to detect the presence of microsatellite instability(MSI)in Marek′s disease(MD)tumors by high-resolution melting(HRM)method.Clinical samples were collected and diagnosed as Marek′s disease by clinical signs,pathological anatomy,histopathology,PCR and sequencing.Muscle,liver,spleen,blood and tumor samples were collected from five affected chickens.DNA was extracted from the samples,and HRM assay was performed by using 15 pairs of primers for amplifying microsatellite DNA sequences.The difference melting curves of the normalization processing were obtained.The differences in the melting curves among samples were compared and analyzed for the presence of MSI in 15 microsatellite markers.The various clinical symptoms and pathological findings of the affected chickens were consistent with MD.The sequence of the viral meq gene had high homology with the virulent strain of MDV prevalent in China in recent years.HRM analysis showed that melting curves of PCR production were significantly different in muscle,liver,spleen,blood,and tumor samples.That is MSI phenomenon and MSI frequencies were different in chickens and microsatellites markers.There were 14,7,5,3 and one microsatellite markers presented MSI in five chickens respectively.The markers MCW0200 and MCW0220 presented MSI in all chickens′samples,however the markers ADL0158 had no MSI phenomenon in all chickens′samples.These microsatellite PCR amplification fragments differ in size were further demonstrated by nondenaturing polyacrylamide gel electrophoresis and sequencing analysis.There was an increase and decrease of microsatellite repeat units,and insertions or deletions of gene fragments in the sequences.The results suggest that the MSI phenomenon in MD tumors can be detected by HRM analysis,and HRM was a sensitive,accurate,simple and high-throughput method.

关 键 词：高分辨率熔解曲线 马立克病 微卫星不稳定性 肿瘤 

分 类 号：S857.4[农业科学—临床兽医学]