Horsburgh Bethany A, Abayasingam Arunasingam, Jenkins Frances, Li Hui, Foster Charles S P, Lloyd Andrew, Coin Lachlan, Rawlinson William, Kelleher Anthony D, van Hal Sebastiaan, Bull Rowena A, Di Giallonardo Francesca
The Kirby Institute, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
Department of Infectious Diseases and Microbiology, Royal Prince Alfred Hospital, Sydney, Australia.
Sci Rep. 2025 Jul 1;15(1):22057. doi: 10.1038/s41598-025-03190-6.
New HIV-1 infections are genotyped as part of standard of care testing to ensure that antiretroviral treatment will be efficacious against the virus. Historically this has been performed by sequencing the pol region of the HIV-1 genome only. The popularity of next-generation sequencing (NGS) methods during the SARS-CoV-2 pandemic has resulted in a shift towards using NGS in diagnostic sequencing, but there remain limited methodologies utilising the strengths of NGS for robust diagnostic sequencing of longer regions of the HIV-1 genome. Given the acceptance and success of tiling PCR methodologies during the SARS-CoV-2 pandemic, we aimed to design and verify a novel tiling PCR method for routine HIV-1 sequencing. A set of tiling PCR primers was designed to amplify the 5' half of HIV-1 in six overlapping segments of 1,000 bp in only two PCR reactions. The assay can move from sample to sequencer in under a day. The tiling PCR was able to generate HIV-1 sequences from 90 (100%) samples in a comparison panel, and complete protease-reverse transcriptase and integrase regions were amplified in > 90% of samples with a viral load > 5000 copies/mL. Seven additional drug resistance mutations were identified when using this novel method. As such, this novel designer tiling PCR is a promising method for the routine NGS-based diagnostic sequencing of HIV-1.
对新的HIV-1感染进行基因分型是标准护理检测的一部分，以确保抗逆转录病毒治疗对该病毒有效。从历史上看，这仅通过对HIV-1基因组的pol区域进行测序来完成。在SARS-CoV-2大流行期间，下一代测序（NGS）方法的普及导致了诊断测序向使用NGS的转变，但利用NGS优势对HIV-1基因组更长区域进行可靠诊断测序的方法仍然有限。鉴于在SARS-CoV-2大流行期间平铺式PCR方法已被接受并取得成功，我们旨在设计并验证一种用于常规HIV-1测序的新型平铺式PCR方法。设计了一组平铺式PCR引物，仅通过两个PCR反应，以1000 bp的六个重叠片段扩增HIV-1的5'半段。该检测方法可在一天内从样本转移到测序仪。在一个比较样本组中，平铺式PCR能够从90个（100%）样本中生成HIV-1序列，并且在病毒载量>5000拷贝/mL的样本中，>90%的样本扩增出了完整的蛋白酶-逆转录酶和整合酶区域。使用这种新方法时，还鉴定出了另外七个耐药突变。因此，这种新型设计的平铺式PCR是一种有前景的方法，可用于基于NGS的HIV-1常规诊断测序。
