In addition, they require the use of gel electrophoresis to detect amplified products, which is long and tedious. Real-time PCR assays developed for the rapid detection of Xcc [4, 8] have the drawback of requiring an expensive thermal cycler with
a fluorescence detector. Loop-mediated isothermal amplification (LAMP) is a recent DNA amplification technique that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions . LAMP is based on the principle of autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase, for the detection of a specific DNA sequence . The technique uses four to six primers that recognize six to eight regions of the target DNA and provides very high specificity [9, 10]. The technique can be carried out PS-341 nmr under FG-4592 cell line isothermal conditions ranging between 60 and 65°C and produces large amounts of DNA . The reaction shows high tolerance to biological Elafibranor in vitro contaminants ,
which can help to avoid false negative results due to the inactivation of the enzyme, a common problem in PCR. Although LAMP amplification products can also be detected by gel electrophoresis, this long procedure reduces the suitability for field applications. For this reason we used SYBRGreen I, an intercalating DNA dye, and a generic lateral flow dipstick (LFD) device to detect the positive amplification by simple visual inspection, as described previously [12–20], with potential field application. We optimized the assay for the amplification of a portion of the pthA gene, a well known pathogenicity determinant of CBC-causing Xanthomonas [21–25]. Various LAMP assays for the detection of animal and human pathogens have been developed [20, 26–33], but this technique remains uncommon for bacterial plant pathogens. Here we describe a sensitive,
specific, fast, and simple LAMP assay for the detection of Citrus Bacterial Canker. Atorvastatin Results Reaction conditions were optimized to establish fast and efficient parameters for amplification. Different temperatures, times and the use of loop primers, which have the capacity to accelerate the reaction, were tested . The optimal amplification of the pthA gene fragment was obtained at 65°C for 30 min using loop primers, as shown by agarose gel electrophoresis (Fig. 1). Amplified products exhibited a typical ladder-like pattern. No products were observed in negative control without DNA (Fig. 1). Specificity of the amplification product was confirmed by sequencing of some bands (data not shown). The samples giving positive reaction show a green color with the addition of SYBRGreen I, while the negative control remained orange (Fig. 2). The lateral flow dipstick shows two clear lines for the positive reaction (the lower line is the sample assay band and the upper one is the control line) while the negative reaction shows only the control line (Fig. 2).