TH21884Y - A set of primers and DNA probes for the detection of genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique, and a DNA biosensor dipstick. - Google Patents

Abstract

The primer and DNA probe kit for detecting the genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick can be summarized as follows: 1. It is a simple, convenient, and rapid diagnostic kit, providing results within one hour, suitable for point-of-care field testing. 2. It exhibits high specificity. 3. It is used for detecting the genetic material of the liver fluke F. gigantica.

Claims (6)

1. A set of primers and DNA probes for detecting the genetic material of parasites. The liver fluke Fasciola gigantica was detected using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick containing four primers capable of binding to the genetic material of F. gigantica: primer Fg-1F3 (5’-3’ sequence: GCGATTGCTGGTTTGTTCAA), primer Fg-1B3 (5’-3’ sequence: AGAGGAACGGATTTGTTGC), primer Fg-FIPLab (5’-3’ sequence: biotin-ACTGTTGCGCACGAAAGCCA-TCTGGGTCGACTTCAACCT), and primer Fg-1BIP (5’-3’ sequence: ...). TAGCCTCGTTCCGGCCAACGTTTCGCACAACAGATCAGTCA 2. A primer and DNA probe set for the detection of genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick, as per claim 1, where the optimal conditions for detecting the LAMP product are characterized by a LAMP reaction at a constant temperature of 60°C for 30 minutes, followed by detection of the LAMP product using the DNA biosensor dipstick. 3. A primer and DNA probe set for the detection of genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick, as per Patent 1, in which the DNA probe for detecting F. gigantica infection has a unique design: a FITC labeled probe at the 5’ end for the detection of the LAMP product on the DNA biosensor dipstick with the following base sequence: 5’-FITC-CAGATTCGTA-3’. 4. The primer and DNA probe kit for the detection of genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick, as per claim 1, consists of the following main components for the reaction: 0.2 micromolar each of primers FG-1F3 and FG-1B3; 2 micromolar each of primers FG-FIP LAP and FG-1BIP; 0.32 units of BST polymerase enzyme; and buffer solution. For a 1x dilution of the enzyme, use 1.4 millimolar (mM) deoxynucleotide triphosphate (dNTPs), 8 millimolar (mM) magnesium sulfate (MgSO4), 0.8 molar (M) betaine, and water (dH2O). 5. The primer and DNA probe set for the detection of genetic material of the liver fluke Fasciola gigantica using the LAMP (loop-mediated isothermal amplification) technique and the DNA biosensor dipstick, as per claim 1, has the unique characteristic of not requiring a thermocycler. 6. A primer and DNA probe set for the detection of genetic material of the liver fluke Fasciola igantica using the LAMP (loop-mediated isothermal amplification) technique and a DNA biosensor dipstick, as per claim 1, for the diagnosis of F. gigantica in clinical samples, namely stool.