One of the great challenges in human immunodeficiency virus (HIV) diagnosis and prevention today is to develop technologies for early and direct detection of HIV nucleic acid sequences in patient tissue or blood samples. Enzymatic amplification of conserved sequences of the HIV-1 genome by PCR has been the subject of a large number of studies. However, the direct biochemical test used for PCR-based molecular diagnosis of HIV-1 infection is usually time consuming, is not quantitative and requires molecular biology facilities. So, direct detection methods that eliminate the requirement for a PCR step could afford faster and simpler devices that can be used outside of a laboratory. Here, a convenient, universal, colorimetric, nucleic acid-responsive detection system that uses two short peptide nucleic acids (PNAs) is demonstrated for the ultra-high sensitive detection of HIV-1 gag DNA and RNA on a 96-well plate based on the sandwich-hybridization strategy. This protocol eliminates the requirement for a PCR step. Using PNA probes instead of traditional DNAs can improve the detection devices for the following reasons: resistance to degradation by enzymes, increased sequence specificity to complementary DNA and RNA, and higher stability when bound with complementary DNA or RNA. Furthermore, the design of a 3-cyclopentane modified surface probe and a 16-biotin containing reporter probe impart extraordinarily high sensitivity. This sandwich-hybridization assay is convenient, universal and colorimetric with a qualitative detection limit of 6 molecules for both of HIV-1 gag DNA and RNA, and a quantitative detection limit of 576 molecules for HIV-1 gag DNA, 493 molecules for HIV-1 gag RNA. These properties should make this device suitable for early detection of HIV virus in remote settings. In principle, this assay can also be used to detect any kind of infectious disease by simply changing the PNA sequences of the specific probe.

Rational design of functional peptide nucleic acids for the direct and ultrahigh sensitive detection of HIV-1 DNA and RNA with a sandwich-hybridization assay

244th National Meeting (2012)

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