摘要
目的建立一种检测甲型H1N1流感病毒耐药基因单碱基突变的滚环扩增技术(rolling cycle amplification,RCA)。方法以甲型H1N1流感病毒耐药基因M2基因和NA基因为研究对象,设计检测该基因突变位点的环化探针,环化探针通过与发生单碱基突变的基因特异性结合并被连接成闭合环状,进行滚环扩增后从而特异性地检测单碱基突变。讨论用于该检测的RCA技术的合适反应条件,通过对人工合成的野生型和突变型靶序列检测,确定该方法的特异性和灵敏度。最后通过对临床标本的检测及与测序结果比对,验证该方法的准确性。结果检测单碱基突变需要严格的反应条件,采用热循环连接法和提高连接温度(65℃),保证了检测特异性。通过对含有不同浓度突变靶序列标本的检测确定了该方法能检测出最低1%含量的突变株。RCA技术检测结果与测序方法结果一致。结论成功建立检测甲型H1N1流感病毒耐药基因单碱基突变的RCA技术。
Objective To establish a rolling circle amplification（RCA） method for detection of single base mutations of influenza A H1N1 resistance genes.Methods M2 and NA resistance gene of H1N1 were target gene in this research.Two circularizable probes（C-probe） were designed to detect these genes.C-probes were ligated to closed rings by DNA ligase when they specifically bond to target genes which had single base mutation.After the rolling circle amplification,the single base mutations were specifically detected.Optimal conditions for RCA method were studied,and the specificity and sensitivity of the method were determined by testing wild and mutation type artificial template.The specimens from clinic were detected and compared with sequencing method to confine its accuracy.Results Strict reaction conditions were needed for detection of single base mutation.Adopting heat cycle ligation and increasing the temperature（65 ℃） could make sure ligation fidelity.Through series detection of the target sequences with different concentrations,the lowest level of mutations detected by RCA was 1% of the total.RCA detection results were consistent with the sequencing method.Conclusion RCA technique is successfully used to detect single base mutation of influenza A H1N1 resistance genes.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第2期177-181,共5页
Journal of Third Military Medical University
基金
国家高技术研究发展计划(863计划
2007AA02Z416)
国家自然科学基金(30400107
81071428)~~
关键词
滚环扩增
甲型H1N1流感病毒
单碱基突变
耐药
rolling circle amplification
influenza A H1N1
single base mutation
resistance
