多引物小型集合HIV核酸检测技术的建立及在男男性接触者急性感染检测中应用

Development of multiple primer, HIV mini-pool NAT and its application in detecting acute infection of MSM

目的 建立结合多重反转录巢式PCR技术检测HIV RNA的小型集合NAT,用于MSM人群急性感染的筛查诊断.方法 利用2008年10月至2009年3月期间从3名HIV窗口期感染者、30名HIV慢性感染者、97名健康人采集冻存的EDTA抗凝血浆建立小型集合NAT.将10份待测标本混合组成1个集合,离心富集病毒,提取RNA 针对HXB2的nt5783-nt6228和nt1235-nt2012区分别设计2对引物 采用多重RT-PCR、巢式PCR扩增2个目标区的核酸片段,对结果阳性的集合中标本进一步分别检测.确定小型集合NAT的灵敏度,并进行验证.应用建立的方法对1 005份与上述标本同一时期收集的MSM人群的HIV抗体阴性血浆进行检测.结果 （1）成功扩增出目标区的2条特异性片段,灵敏度为162拷贝/ml （2）对3份HIV窗口期感染者标本用小型集合NAT方法盲法验证的结果和预期一致 （3）用多重RT-PCR和巢式PCR对30份HIV抗体阳性标本检测,阳性率100%（30/30） （4）发现1 005份血浆标本中有1份为HIV RNA阳性,追踪随访发现HIV-1抗体转阳.结论 本研究中建立多引物小型集合RT-PCR、巢式PCR结合的小型集合NAT的敏感性好,可用于MSM人群HIVG感染窗口期筛查检测.

Objective To establish a mini-pool nucleic acid testing （NAT） assay using multiplex RT-nested PCR for the detection of HIV RNA, and apply it in screening for acute HIV infection among MSM. Methods Frozen EDTA plasma samples collected between Oct. 2008 and Mar. 2009 from 3 HIV infectors during window-period, a total of 30 HIV chronically infected individuals and 97 healthy subjects were used to develop the NAT assay. Plasma samples from 10 cases were pooled into one tube and centrifuged at high speed for the collection of viruses. HIV RNA was extracted. Two pairs of primers were designed according to two conserved regions of HIV RNA （ HXB2 nt 5783-nt 6228 and nt 1235-nt 2012）.Multiplex RT-PCR and nested PCR were performed. Individual NAT-reactive samples were confirmed by commercially available NAT assays. The sensitivity and performance efficacy were also evaluated. The assay was then applied to 1 005 plasma specimens from MSM with negative or uncertain HIV antibody test results.These were collected in the same period as the other samples. Results （ 1 ） Two fragments of HIV were amplified successfully with the low detection limit of 162 copies/ml plasma （2） Results of the mini-pool HIV NAT validation with samples from 3 HIV infectors during window-period were consistent with the expected values （3） All 30 plasma samples from MSM with positive HIV antibody, which were tested by multiplex RT nested PCR, were found to be HIV RNA positive （4） One out of 1 005 plasma samples was found to be HIV RNA positive, for this case acute infection was followed-up and sero-conversion was found. Conclusion Mini-pool NAT has good sensitivity, and may be applied to screening HIV RNA among MSM during window-period.