A set of primers and DNA probes specific to the ITS1 region for the detection of the small intestinal fluke Centrocestus formosanus, along with a detection method using Loop-mediated isothermal amplification (LAMP) technique combined with a Lateral Flow Dipstick (LFD).

Abstract

------09 / 10 / 2568------(OCR) OCR 09WP This primer and DNA probe set is specific to the ITS1 region for the detection of the small intestinal fluke Centrocestus formosanus. The detection method uses Loop-mediated isothermal amplification (LAMP) technique combined with a Lateral Flow Dipstick (LFD). This method is highly specific and accurate, easy to use, and fast, providing results within 1 hour and 30 minutes. It can be used to detect Centrocestus formosanus in economically important fish.

Claims

------30 / 04 / 2569------(OCR) OCR 10KL1. A set of primers and DNA probes specific to the ITS1 region for the detection of the small intestinal fluke Centrocestus formosanus using the loop-mediated technique. Loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) consisting of four primers and one DNA probe specific to the ITS1 region of the small intestinal fluke Centrocestus formosanus, with the following nucleotide sequences: Primer CF-ITS1-F3 5'-GCCATTTGACGACCAACATG-3', Primer CF-ITS1-B3 5'-CACATCCCGTCACTCCCA-3', Primer CF-ITS1-FIP. (CF-ITS1-FIP) 5'-Biotin-CAGGTATAGCCTGGAGATGGCGTTTTTCGGTGCATTTTGCAGTCA-3' Primer CF-ITS1-BIP (CF-ITS1-BIP) 5'-ACAGTOGACCTTTCGACAGGTTTTTCCACCAAAGGTAGCAAGCAC-3' Probe CF-ITS1-Probe (CF-ITS1-Probe) 5'-FAM-ACTGCCTAAGGTGAAGC-3' 2.A primer and DNA probe specific to the ITS1 region for the detection of the small intestinal fluke Centrocestus formosanus using the LAMP technique in conjunction with a colorimetric detector (LFD), as per Patent 1, where the DNA probe is characterized by being labeled with FAM at the 5' end for the detection of LAMP products on the LFD. The sequence is as follows: 5'-FAM-ACTGCCTAAGGTGAAGC-3' 3. Method for detecting the small intestinal fluke Centrocestus formosanus using the LAMP technique combined with a colorimetric strip detector (LFD) using a primer set and DNA probe according to claim 1 or 2, which consists of the following steps: a. Amplification of the genetic material of the submitted sample using the LAMP technique with a primer set. The optimal conditions for the LAMP reaction are a temperature of 60-68 degrees Celsius for 30-60 minutes. b.The detection of the LAMP product on a colorimetric strip (LFD) is performed by mixing the DNA probe with the LAMP product and then hybridizing it. The mixture is then aspirated into a buffer solution, the LFD is immersed in the solution, and the result is read from the appearance of a colored band.

4. Method for the detection of the small intestinal fluke Centrocestus formosanus using the LAMP technique in conjunction with a colorimetric strip (LFD) according to claim 3, where 12.5 microliters (µL) of LAMP reaction consists of the following main components: The outer primers are CF-ITS1-F3 and CF-ITS1-B3, each at 0.2 micromolar; the inner primers are CF-ITS1-FIP and CF-ITS1-BIP, each at 1.5 micromolar; deoxynucleotide triphosphate (dNTPs) at 1.4 millimolar; and betaine at 1.5 micromolar.0 molar (M), magnesium sulfate (MgSO4) 3 millimolar (millimolar), Bst DNA polymerase enzyme 8 units (U), 10X isothermal amplification buffer, and ultrapure water.

5. A method for the detection of Centrocestus formosanus using the LAMP technique combined with a colorimetric detector (LFD) according to patent 3 or 4, which is used for the detection of Centrocestus formosanus in commercially important fish samples. ------09 / 10 / 2568------(OCR) OCR 09WP1.A set of primers and DNA probes specific to the ITS1 region for the detection of the small intestinal fluke Centrocestus formosanus, along with a detection method using Loop-mediated isothermal amplification (LAMP) technique combined with a Lateral Flow Distick (LFD). The set includes four primers and one DNA probe specific to the ITS1 region of Centrocestus formosanus, with the following nucleotide sequences: Primer CF-ITS1-F3 (CF-ITS1-F3)5'-GCCATTTGACGACCAACATG-3', Primer CF-ITS1-B3... (CF-ITS1-B3)5'-CACATCCCGTCACTCCCA-3'primerCF-ITS1-FIP (CF-ITS1-FIP)5'-Biotin-CAGGTATAGCCTGGAGATGGCGTTTTTCGGTGCATTTTGCAGTCA-3'primerCF-ITS1-BIP (CF-ITS1-BIP)5-ACAGTCGACCTTTCGACAGGGTTTTTCCACCAAAGGTAGCAAGCAC-3'probeCF-ITS1-Probe (CF-ITS1-Probe)5'-FAM-ACTGCCTAAGGTGAAGC-3'2.A primer and DNA probe specific to the ITS1 region for the detection of Centrocestus formosanus using the LAMP technique combined with a Long-Range Finding Device (LFD) according to Patent 1, where the DNA probe is characterized by being labeled with FAM at the 5' end for the detection of the LAMP product on the LFD, with the sequence 5'-FAM-ACTGCCTAAGGTGAAGC-3'3. The procedure for the detection of Centrocestus formosanus using the LAMP technique combined with a LFD, using the primer and DNA probe set according to Patent 1 or 2, consists of the following steps: a. The amplification of genetic material in submitted samples using the LAMP technique involves employing a set of primers. The optimal conditions for the LAMP reaction are a temperature of 60-68 degrees Celsius for a duration of 30-60 minutes.The detection of the LAMP product on a colorimetric strip (LFD) is performed by mixing the DNA probe with the LAMP product and then hybridizing it. The mixture is then aspirated into a buffer solution, the LFD is immersed in the solution, and the result is read from the appearance of a color band.

4. A method for detecting the small intestinal fluke Centrocestus formosanus using the LAMP technique in conjunction with a colorimetric strip (LFD) according to claim 3, where 12.5 microliters (µL) of LAMP reaction consists of the following main components: The outer primers are CF-ITS1-F3 and CF-ITS1-B3, each at 0.2 micromolar (mM); the inner primers are CF-ITS1-FIP and CF-ITS1-BIP, each at 1.5 micromolar (mM); deoxynucleotide triphosphate (dNTPs) at 1.4 millimolar (mM); and betaine at 1.5 micromolar (mM).0 molar (M), magnesium sulfate (MgSO4) 3 millimolar (mM), Bst DNA polymerase enzyme 8 units (U), 10X isothermal amplification buffer, and ultrapure water.

5. A method for detecting the small intestinal fluke Centrocestus formosanus using the LAMP technique combined with a colorimetric strip detector (LFD) according to patent 3 or 4, which is used for the detection of Centrocestus formosanus in economic fish samples.

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