| Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification approach that was firstly reported in2000. The mechanism of LAMP is designing four specific primers according to six separate conservative regions of the target genes, including a couple of internal primers (FIP and BIP) and a couple of external primers (F3and B3), and combining the Bst DNA polymerase at a constant temperature (63℃) for30-60min. At a given temperature, LAMP achieves amplification only by one step but with extreme high efficiency. It can reach109～1010times amplification in30-60min. Additionally, LAMP takes advantages on the observation of the results. When the experiment is completed, white precipitation will be observed. If calcein are added initially, it is feasible to determine the termination via the color change. The appearance of yellow green color indicates the amplification occurred, in which the electrophoresis is non-necessarily any more. Due to its high efficiency, simplicity, rapidity, high specificity and the result of LAMP is easily detected by naked eyes, LAMP has been widely applied in the qualitative and quantitative detection of viruses, bacteria and clinical diagnosis of disease. In this paper, we have introduced the principle of LAMP and its application for detection of Ectromelia virus. By comparing the results to those obtained by PCR, a novel detection method for Ectromelia virus based LAMP approach is developed herein.Designing several primes based on the conservative genes of the Ectromelia virus, then screened the optimal primer in LAMP experiments by monitoring the process with real time turbidity meter, where the temperature was set to be63℃and the reaction time was1hour to measure the sensitive and specific parameters of the primers to Ectromelia virus. In comparing the results obtained by PCR, one could conclude the optimal sensitivity of two methods. The primer selection experiments choosed a couple of primers that designed based on Crmd gene, and then measured the specificity of the primers. The selected primers only reacted with Ectromelia virus rapidly and reached the amplification summit in5minutes without any reaction of other viruses that are susceptible to mice. In the sensitivity experiments, the concentration of Ectromelia virus DNA was diluted in the range of100to10-6for7samples and the detection limit was5.3x10-4ng/ul, which was much higher than that of PCR. In order to simplify the observation of results, calcein indicator was added to the Ectromelia virus LAMP experiment. Comparing to negative control, the amplification reaction appeared with a yellow green fluorescent, which was visible by the naked eye and further experiment by agarose gel electrophoresis showed the continuous stripe.In order to enhance the reaction rate, a novel loop primer was designed on basis of original LAMP, therefore a modified LAMP experiment was developed in comparing to the standard one. The loop primer, which was a primer by attaching a compensative sequence loop primer, such as loop primer F (LF), loop primer B (LB), to the end of the single strand regime of dumbbell-liked5’structure (the regime between B1to B2, F1to F2). Thus, the start points of the reaction were increased and subsequently the rate was enhanced. Though only one loop primer was designed for Ectromelia virus herein, the enhancement of the reaction rate was significantly.During the regression experiment, BALB/c mice and BALB/c nude mice were used, virus was diluted and divided into6groups and each group contains BALB/c mice and BALB/C nude mice. Take the serum and tissues of the mice and extract the viral DNA. The results indicated that all samples were feasible to amplify with LAMP experiment, and indirect ELISA experiment indicated that BALB/c mice were positive but BALB/c nude mice were negative. In conclusion, with the phenomenon of false positive by indirect ELISA, LAMP is more convenient and intuitive to detect Ectromelia virus. |
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