ABSTRACT
ObjectiveThe traditional detection method of recombinase polymerase amplificaton (RPA) is not suitable for rapid field detection due to the complicated operation and other factors. Taking the detection of hepatitis b virus (HBV) nucleic acid as an example, it established a detection method of HBV nucleic acid isothermal amplification based on recombinase polymerase amplificaton (RPA) and designed a matching visual detection device of RPA product.MethodsFirstly, a RPA product detection device was designed, which can be used to collect images by taking photos of mobile phones and visually interpret the detection results. Secondly, RPA primers and probes were designed according to the design of HBV gene conserved sequence. Amplification efficiency of each primer pairs were compared though monitoring the RPA reaction of real-time fluorescence curve to screen the best primers and optimize the optimal reaction conditions. Visual detection sensitivity was investigated by using artificial synthesis of HBV target plasmid, and was investigated the specificity of the method by the detection of synthetic plasmid containing hepatitis c virus (HCV), human immunodeficiency virus, treponema pallidum, influenza virus, human papilloma virus DNA fragment. Thirdly, the feasibility of RPA product visualization detection device was verified by comparing with the real-time fluorescence amplification curve. Finally, RPA visual detection was performed on 20 serum DNA samples detected by real-time fluorescence PCR to evaluate the applicability of this method to the detection of actual clinical samples.ResultsThe visual detection device of RPA product was used to realize the negative and positive signals that could be detected by mobile phone photography and visual observation. The visual detection method of HBV nucleic acid RPA amplification could realize the visual detection of DNA targets as low as 1-10 copies of HBV within 30 min at 39 ℃ and had good specificity. The test results of 20 serum DNA samples were completely consistent with those of the commercially available qPCR kit, which preliminarily verified the practicability of the method and the device.ConclusionCombined the established HBV-RPA amplification system with the RPA product visualized detection device, it would be expected to develop a low-cost rapid visualization screening technology platform for HBV nucleic acid in blood.
ABSTRACT
Objective To understand the level and distribution of antibody F1 against plague in population of Ningxia natural plague foci in 2007 and 2008. Methods Seven hundred and eighteen blood samples were collected in five major cities and counties of natural plague foci, and 475 blood samples were collected in nonplague area as control group. Conventional indirect hemagglutination, colloidal gold test, and enzyme-linked immunoassay were employed to test the antibody. If the result was tested positive by more than two methods used then the result was defined as positive. Antibody titer that did not reach the positive standard was defined as suspected samples. Results A total of 718 serum samples were tested, the results showed that 9 samples were positive (antibody titer was 1:16 - 1:64), the positive rate was 1.25%(9/718), suspected samples was 28, the detection rate was 3.90%(28/718). Four hundred and seventy-five serum samples in the non-plague area were all negative by the three methods. There was a significant difference of antibody F1 positive rate between residents in historical epidemic area and history nonepidemic area(χ2 = 4.44, P< 0.05). There was no statistical significance of the positive rate[1.25%(9/718), 1.25%(9/718),2.51%(18/718)]among the three methods used(χ2 = 1.91, P> 0.05). Conclusion There still exists a certain proportion of Fl antibody positive people in Ningxia natural plague foci, and these people are distributed in areas where several animal plague prevalent in recent years.