采用单管一步法逆转录聚合酶链反应（RT PCR）及基于TaqMan探针的实时荧光定量PCR法对HIV-1 RNA载量进行定量分析。
Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.
作者信息
Watanaveeradej Veerachai, Sirirattanaphoomee Sudalak, Chantratita Wasun, Nitayaphan Sorachai, Viputtikul Kwanjai, Kerdpanich Angkool, Samakoses Rudiwilai, Simasathien Sriluk
机构信息
Deparment of Pediatrics, Phramongkutklao Hospital, Bangkok, Thailand.
出版信息
J Med Assoc Thai. 2005 Nov;88 Suppl 3:S206-13.
OBJECTIVES
To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries.
MATERIAL AND METHOD
An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR.
RESULTS
IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR.
CONCLUSION
A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.
目的
开发一种成本较低的检测方法来计算HIV-1病毒载量，以供资源有限的国家使用。
材料与方法
采用基于TaqMan探针的实时PCR技术开发了一种内部单管一步病毒载量检测方法（IOVA）。引物和探针是根据所有HIV亚型序列的保守区域设计的。通过对不同稀释度的参考病毒生成标准曲线。将IOVA应用于105份HIV阳性样本和25份HIV阴性样本，并与罗氏AMPLICLOR的检测结果进行比较。
结果
IOVA检测样本中的HIV RNA含量范围为125至2×10⁶拷贝/毫升。批内和批间变异系数范围为0.68%至7.89%。灵敏度、特异性、阳性预测值和阴性预测值分别为92%、100%、100%和79.5%。平行定量分析显示IOVA与AMPLICOR之间具有高度相关性（r = 0.95）。
结论
开发并验证了一种新的HIV-1病毒载量检测方法。该方法可靠且成本较低。
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