Ou C Y, McDonough S H, Cabanas D, Ryder T B, Harper M, Moore J, Schochetman G
Division of HIV/AIDS, Centers for Disease Control, Atlanta, GA.
AIDS Res Hum Retroviruses. 1990 Nov;6(11):1323-9. doi: 10.1089/aid.1990.6.1323.
A hybridization protection assay (HPA) that uses acridinium ester (AE) labeled oligonucleotide probes which are specific for a conserved gag gene region of human immunodeficiency virus type 1 (HIV-1) was developed to measure the amount of HIV-1 nucleic acid. Hybridization of the single-stranded probes with their target HIV-1 sequences protected the chemiluminescent AE group from subsequent alkaline hydrolysis. The chemiluminescence from the residual AE could be easily quantitated in a luminometer. The entire process comprising template dissociation, hybridization, alkaline hydrolysis, and chemiluminescence measurement can be completed in less than one hour and does not require the separation of hybridized probe from unhybridized probe. We demonstrated that HPA could quantitatively measure the amount of DNA amplified by polymerase chain reaction. A comparative study using amplified DNA from the peripheral blood mononuclear cells (PBMC) of HIV seropositive and seronegative persons showed that HPA was as sensitive as the previous methods using 32P-labeled DNA probes.
开发了一种杂交保护分析（HPA）方法，该方法使用吖啶酯（AE）标记的寡核苷酸探针，这些探针特异性针对人类免疫缺陷病毒1型（HIV-1）的保守gag基因区域，用于测量HIV-1核酸的量。单链探针与其靶标HIV-1序列的杂交保护了化学发光的AE基团不被随后的碱水解。残留AE的化学发光可以在发光计中轻松定量。包括模板解离、杂交、碱水解和化学发光测量的整个过程可以在不到一小时内完成，并且不需要将杂交探针与未杂交探针分离。我们证明HPA可以定量测量通过聚合酶链反应扩增的DNA量。一项使用HIV血清阳性和血清阴性者外周血单核细胞（PBMC）扩增DNA的比较研究表明，HPA与先前使用32P标记DNA探针的方法一样灵敏。
