Establishment of real-time fluorescence loop-mediated isothermal amplification assay for rapid detec-tion of HIV-1 DNA / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish a rapid and high sensitive assay of real-time fluorescence loop-mediated isothermal amplification assay for clinical detection of HIV-1 DNA.Methods Four loop primers were constructed for loop-mediated isothermal amplification ( LAMP) assay based on six conserved regions selected from HIV gene sequence .SYBR Green Ⅰdye was added into the established LAMP assay and its specificity and sensitivity were evaluated .Results The real-time fluorescence LAMP assay for the detection of HIV-1 DNA was successfully established .It was used for the detection of HIV-1 DNA among 200 patients with HIV infection, of which 195 cases were positive.Moreover, 50 healthy subjects were found HIV-1 DNA negative tested by the real-time fluorescence LAMP assay .Quantitative testing for HIV-1 DNA showed that the lowest and the highest detectable amount were 51 copies/ml and 8.21×106 copies/ml respectively, and the average amount was 5.78×105 copies/ml.HIV viral loads ranging from 1×105 to 10×105 were detected in 162 of 200 patients (83.08%).The ten times dilution method showed that the lowest detection limit of the assay was 50 copies/ml.The crossover experiment indicated that the specificity of the assay was 100%as none of HBV-DNA, HSV-DNA and HCV-RNA was determined by the assay .Conclusion The present study shows that 97.5%of the patients with HIV infection are confirmed HIV-1 DNA positive by the real-time fluorescence LAMP assay , suggesting that the real-time fluorescence LAMP assay is a rapid and sensi-tive assay with high specificity and could be applied for clinical detection of HIV-1 DNA.