Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes

作者：Li Peilin^*; Ruel Theodore; Fujimoto Katsuya; Hatano Hiroyu; Yukl Steven; Eller Leigh Anne; Liegler Teri; Kamya Moses; Gassasira Anne; Dorsey Grant; Rosenthal Philip J; Havlir Diane V; Wong Joseph K

来源：Journal of Virological Methods, 2010, 170(1-2): 115-120.

DOI：10.1016/j.jviromet.2010.09.011

• HIV
• Taqman PCR
• HIV subtypes
• Dried blood spots
• Locked Nucleic Acid

There remains a need for sensitive and cost-effective assays to monitor therapy in human immunodeficiency virus type-1 (HIV-1) infection. However, the genetic diversity of HIV poses difficulties for traditional real-time PCR assays that require long oligonucleotides probes. LNA (TM) probes may be useful in overcoming these limits to traditional probe design. A new application of LNA (TM) chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA (TM) probe that matches the majority of HIV-1 sequences in the Los Alamos database, exhibited a wide dynamic range (10(1)-10(7) copies of HIV DNA), high sensitivity (limit of detection of 1 copy of HIV DNA in 10(5) cells), and broad applicability to a range of HIV-1 subtypes (including A, B. C, D. F, H, B/C, and A/E CRFs). Using the LNA (TM) probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naive HIV-1 positive Ugandan children, and HIV DNA levels significantly correlated with viral RNA levels in plasma (r = 0.765, p < 0.0001). This approach to Taqman probe design should be explored further for use in diagnosis and monitoring of HIV in resource-limited settings, especially where several subtypes co-circulate. Published by Elsevier B.V.

• 出版日期2010-12