A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load

被引:130
作者
Malnati, Mauro S. [2 ]
Scarlatti, Gabriella [1 ]
Gatto, Francesca [2 ]
Salvatori, Francesca [1 ]
Cassina, Giulia [2 ]
Rutigliano, Teresa [2 ]
Volpi, Rosy [1 ]
Lusso, Paolo [2 ]
机构
[1] Ist Sci San Raffaele, Unit Viral Evolut & Transmiss, Dept Biol & Technol Res DIBIT, I-20132 Milan, Italy
[2] Ist Sci San Raffaele, Unit Human Virol, I-20132 Milan, Italy
关键词
D O I
10.1038/nprot.2008.108
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.
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页码:1240 / 1248
页数:9
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