Detection of Phylogenetically Diverse Human Immunodeficiency Virus Type 1 Groups M and O from Plasma by Using Highly Sensitive and Specific Generic Primers

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the “window period.” Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）具有极高的种内遗传多样性，其包含M（主要组，major）、O（偏离组，outlier）两个主要组群，且M组内存在多种env亚型（A至J亚型），这使得设计可覆盖所有已知HIV-1毒株的检测方法极具挑战性。
本研究基于跨膜蛋白gp41的保守免疫优势区域开发了一套通用引物组，该引物组能够可靠扩增来自M组A至H亚型（I、J亚型未获取）的近全长克隆的病毒DNA，最低检测限可达10拷贝/聚合酶链式反应（polymerase chain reaction, PCR）。
该检测方法对多种地理起源、不同亚型的HIV-1 M组毒株以及HIV-1 O组毒株的血浆病毒RNA均具有极高的检测灵敏度。在253份M组血浆标本（亚型A：68份；B：71份；C：19份；D：27份；E：23份；F：33份；G：12份）中，250份（98.8%）可通过gp41 M/O引物组实现扩增。更为关键的是，全部32份（100%）O组血浆样本也可通过该引物组成功扩增。
体外加标实验进一步证实，该检测方法能够可靠检出低至25拷贝/毫升的病毒RNA，且对于血浆病毒载量低于所有商用病毒载量检测方法检出限的HIV-1血清阳性标本，也可呈现阳性信号。此外，对5份血清转换期样本队列的分析表明，该检测方法在“窗口期”内能够高灵敏度地早期检出血浆病毒血症。
综上，这款高灵敏度检测方法可用于临床标本中所有已知HIV-1感染的早期检测，不受其基因型与地理起源的限制。