人偏肺病毒基因芯片检测方法的建立及初步应用

Establishment and Preliminary Application of the Gene Chip Detection Method of Human Pneumosis Virus

目的建立深圳地区人偏肺病毒(human metapneumovirus,hMPV)核苷酸的快速、特异和敏感的基因芯片检测方法,为hMPV检测和诊断提供有效的诊疗手段。方法针对hMPV病毒高度保守区域基因序列,应用引物设计软件Primer5设计hMPV的荧光PCR引物;应用探针设计软件Array Designer4.20设计hMPV的寡核苷酸检测探针,将寡核苷酸探针点样至醛基玻片上制备hMPV基因芯片,并与常规反转录酶-聚合酶链锁反应(RT-PCR)平行比较,对其灵敏度、特异度和重复性,以及用于临床样本的适用性等进行评价。结果 hMPV基因芯片法检测hMPV的特异度为96.23%(230/239),灵敏度可达2.0×10~1个/μl,线性范围为2.0×101~2.0×10~7个/μl,且重复性好;初步检测深圳地区300份临床鼻咽拭子标本,基因芯片法检出率为20.3%(61/300),明显高于常规RT-PCR法的9.7%(29/300),两方法检测的灵敏度之间差异有统计学意义(χ~2=39.205,P<0.05);两种方法检测结果一致性一般(kappa=0.360 7)。结论建立了hMPV基因芯片检测法,基因芯片法检测hMPV具有很高的特异度和灵敏度,且检测线性范围广,为实验室开展hMPV的流行监测和早期诊断提供了新的检测技术。

Objective To establish human metapneumovirus（hMPV）nucleotide rapid,specific and sensitive gene chip detec-tion method,and provide effective diagnostic methods for hMPV detection and diagnosis in shenzhen area.Methods The flu-orescence PCR primer of hMPV was designed for the highly conservative regional gene sequence of hMPV virus.Application of array probe design software designer 4.20 hMPV oligonucleotide detection probe design,oligonucleotide probe sample points to aldehyde slides on the preparation of hMPV gene chip,and parallel compared with conventional reverse tran-scriptase-polymerase chain reaction（RT-PCR）,the sensitivity,specificity and repeatability,and was used to evaluate the clinical applicability of the samples.Results hMPV gene chip method to detect hMPV specificity was 96.23%（230/239）, the sensitivity was 2.0×101/μl,linear range was 2.0×10^1~2.0×107/μl,and the repeatability was good.Initial tests in shenzhen area 300 clinical specimens on a nasopharyngeal swab,gene chip method detection rate was 20.3%（61/300）,sig-nificantly higher than the conventional RT-PCR method 9.7%（29/300）,the difference was statistically significant between the sensitivity of two methods（χ2=39.205,P〈0.05）.The results were consistent in two methods（kappa=0.360 7）.Con-clusion Established hMPV microarray assay,gene chip method to detect hMPV has high specificity and sensitivity,wide linear range and detection.The popularity of hMPV monitoring for laboratory provides a new detecting technology and early diagnosis.