Abstract
Sensitive and specific detection of HIV-related DNA is of great importance for early accurate diagnosis and therapy of HIV-infected patients. Here, we developed a one-step and rapid fluorescence strategy for HIV-related DNA detection based on strand displacement amplification and a Mg2+-dependent DNAzyme reaction. In the presence of target HIV DNA, it can hybridize with template DNA and activate strand displacement amplification to generate numerous DNAzyme sequences. With the introduction of Mg2+, DNAzyme can be activated to circularly cleave the substrate DNA, which leads to the separation of fluorophore reporters from the quenchers, resulting in the recovery of the fluorescence. Under the optimal experimental conditions, the established biosensing method can detect target DNA down to 61 fM with a linear range from 100 fM to 1 nM, and discriminate target DNA from mismatched DNA perfectly. In addition, the developed biosensing strategy was successfully applied to assay target DNA spiked into human serum samples. With the advantages of fast, easy operation and high-performance, this biosensing strategy might be an alternative tool for clinical diagnosis of HIV infection.
Highlights
Human immunode ciency virus (HIV), a retrovirus, can lead to acquired immunode ciency syndrome (AIDS) which destroys the infected patient's immune system.[1]
The feasibility of the SDA reaction was explored by 8% native polyacrylamide gel electrophoresis (PAGE), which was conducted on DYY-6C electrophoresis analyzer (Liuyi Instrument Company, China) in 1Â TBE buffer (90 mM Tris–HCL, 90 mM boric acid, 2 mM EDTA, pH 7.9) at a 120 V constant voltage for 30 min
The template consists of two nucleic acid scaffolds that include recognition site for the target DNA and replication track that yields the nicking domain for Nb.BbvCI and Mg2+-dependent DNAzyme sequence
Summary
Human immunode ciency virus (HIV), a retrovirus, can lead to acquired immunode ciency syndrome (AIDS) which destroys the infected patient's immune system.[1]. Nucleic acid assays have attracted increasing attention because they can prompt for viral infection even in the absence of HIV antibodies.[9] With high sensitivity and speci city, polymerase chain reaction (PCR) has evolved as the most commonly used technique for nucleic acid detection. It is not suitable for short-length DNA detection due to the complexity in primer design.[10] PCR-based methods need thermal cycler and specialized person which limit their use in resource-poor and impoverished areas. The proposed uorescence biosensing strategy presents an excellent platform towards HIV-related DNA analysis, which has great application potential for biomedical research and clinical diagnosis of HIV infection
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