Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes.
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™ probes may be useful in overcoming these limits to traditional probe design. A new application of LNA™ chemistry in a Taqman assay applicable to a wide range of HIV-1 subtypes is described. This assay, based on a 13-mer LNA™ 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™ probe assay, HIV-1 DNA was detected in all dried blood spots (DBS) from treatment naïve 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.
Li
Peilin
P
University of California, San Francisco (UCSF), Department of Medicine, San Francisco, CA 94121, USA. Peilin.li@va.gov
Ruel
Theodore
T
Fujimoto
Katsuya
K
Hatano
Hiroyu
H
Yukl
Steven
S
Eller
Leigh Anne
LA
Liegler
Teri
T
Kamya
Moses
M
Gassasira
Anne
A
Dorsey
Grant
G
Rosenthal
Philip J
PJ
Havlir
Diane V
DV
Wong
Joseph K
JK
D002648:Child; D004279:DNA, Viral / Q000097:blood*; D018740:Genetic Heterogeneity; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D011933:Reagent Kits, Diagnostic / Q000191:economics; D012680:Sensitivity and Specificity; D014454:Uganda
D004279:DNA, Viral; D009841:Oligonucleotides; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; C477371:locked nucleic acid