Journal: The Korean Journal of Parasitology
Article Title: Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)
Figure Lengend Snippet: Detection limit of Azumiobodo hoyamushi 18S rDNA LAMP assays (A). LAMP assays were performed using serial dilutions of A. hoyamushi genomic DNA (1 ng to 1 fg per reaction). Distilled water was used as a negative control. LAMP products were visualized by gel electrophoresis (B) and using Loopamp® fluorescent detection reagent (FD) (C). (B, C) Lane M, 100-bp DNA marker; lane 1, 1 ng; lane 2, 100 pg; lane 3, 10 pg; lane 4, 1 pg; lane 5, 100 fg; lane 6, 10 fg; lane 7, 1 fg of A. hoyamushi genomic DNA; lane 8, distilled water; and lane 9, LAMP product after Mbo I digestion. (D-E) A. hoyamushi at a density of 1×10 3 parasites/µl was serially diluted and tested (D) using the LAMP assay (D) and by PCR (E) using F3 and B3 primers. Lane M, 100-bp DNA marker; lane 1, 1,000; lane 2, 100; lane 3, 10; lane 4, 1; lane 5, 0.1; lane 6, 0.01 of parasites per reaction; lane 7, distilled water. A. hoyamushi genomic DNA was prepared using DNeasy tissue kits (Qiagen) from in vitro cultured A. hoyamushi species [ 9 ] which were kindly provided by Dr. Kyung Il Park (Kunsan National University, Gunsan, Korea).
Article Snippet: A. hoyamushi LAMP was performed for 90 min at 64℃ in a 25 µl mixture containing 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, 20 pmol each of LF and LB, 1.4 mM of deoxynucleoside triphosphates, 0.8 M betaine, and 1 µl of Bst DNA polymerase (NEB, Beverly, Massachusetts, USA) in 2.5 µl of buffer [20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , and 0.1% Tween 20], and 1 µl of template DNA in a Loopamp real-time turbidimeter (Realoop-30; Eiken Chemical Co., Tokyo, Japan).
Techniques: Negative Control, Nucleic Acid Electrophoresis, Marker, Lamp Assay, Polymerase Chain Reaction, In Vitro, Cell Culture