TH12062Y - A method for detecting pathogenic Streptococcus iniae bacteria in fish. - Google Patents

Abstract

Revised 19/2/59. This invention develops a method for detecting S. iniae bacteria in fish using a color-changing lamp technique involving hydroxynaptal blue mixed into the lamp reaction solution before the reaction begins. The process is a single-step reaction by designing primers specific to eight base sequences on the superoxide dismutase (soc/A) gene of S. iniae bacteria, and determining the optimal reaction composition and conditions. This results in a specific, rapid, and user-friendly method for diagnosing pathogenic bacteria in economically important fish, eliminating the need for complex instruments. ------------------------------------------------------------------------------------------------------ New Request (Improved): This invention develops a method for detecting S. iniae bacteria in fish using a color-changing lamp technique involving hydroxynaptal blue mixed into the lamp reaction solution before the reaction begins. The process is a single-step reaction by designing primers specific to eight base sequences on the superoxide dismutase (sodA) gene of S. iniae bacteria, and determining the optimal reaction composition and conditions. This makes the diagnostic methods for pathogenic bacteria in economically important fish more specific, rapid, and user-friendly, without the need for complex equipment.

Claims (2)

Revised 19/2/59 1. The procedure for detecting Streptococcus iniae bacteria causing disease in fish consists of the following steps: a. Take a 50 mg tissue sample from the desired fish organ and crush it with 2 molar DNA extraction solution and 250 µL of guanidine thiocyanate solution, or add more until the extraction solution can thoroughly crush the sample. Alternatively, take a 100 µL blood sample without anticoagulant and crush it with 200 µL of DNA extraction solution (0.025 normal sodium hydroxide solution and 0.0125% sodium dodecyl sulfate solution), or add more until the DNA extraction solution... a. The sample can be ground until homogeneous. b. Boil the test tube from step a at 95 degrees Celsius for 5 minutes. Then, immerse the test tube in ice for another 5 minutes. Tissue precipitate will be observed settling to the bottom of the test tube. c. Dilute the DNA solution 50-fold to obtain the appropriate amount of DNA for the reaction. Mix 10 microliters of the clear solution with 490 microliters of distilled water. d. Add 2 microliters of the DNA solution extracted from the fish sample from step c to the LAMP solution. Composed of: - Outer primer F3, base sequence 5’ –TAATGGTGGCGGACACTT-3’ 0.2 micromolar - Outer primer B3, base sequence 5’ – AAGTAATTTCAAGACTTCCTTCT-3’ 0.2 micromolar - Inner primer FIP, base sequence 5’ – TGCACTTGCAACTTCTTTTGTTACTTTTTAATCATGCTTTGTTCTGGGA-3’ 2.0 micromolar - Inner primer BIP, base sequence 5’ – AAGAACAATTTGCAGCAGCAGCTTTTAACTTCTAACCAAGCCCA-3’ 2.0 micromolar - Loop primer LF, base sequence 5’ –ACTGGCCGTTTTGGTTCTGGT-3’ 2.0 micromolar - Loop primer FB, base sequence 5’ –GACCAAGCTTTTCCATC-3’ 2.0 micromolar - Buffer at 1x concentration (Tris-HCL 20 millimolar, (NH4)2SO4 10 millimolar, KCl 10 millimolar, MgSO4 2 millimolar, Triton X-100 0.1 percent, pH 8.8) - Magnesium sulfate solution 4.0 millimolar - Betaine 0.5 molar - dNTPs 1.4 millimolar - Bst enzyme DNA polymerase 8 units - Hydroxynaptal blue solution 120 micromolar. E. Heat all components to 63 °C for 45 minutes. F. Read results visually immediately. In samples infected with Streptococcus iniae bacteria, the reaction solution will change from purple (negative test result) to blue (positive test result). ------------------------------------------------------ New request updated. 1. The procedure for detecting pathogenic Streptococcus iniae bacteria in fish consists of the following steps: 1.1 Preparation of DNA solution extracted from the fish sample to be tested: a. Cut approximately 50 milligrams of tissue from the organ of the fish to be tested. Grind it with 2 molar DNA extraction solution (250 microliters), or add more solution until the extraction solution can thoroughly grind the sample. b. Or, take a 100 microliter blood sample without anticoagulant and grind it with 200 microliters of DNA extraction solution, or add more solution until the extraction solution can thoroughly grind the sample. c. Heat the test tube from step a or b at 95 degrees Celsius for 5 minutes. Then, immerse the test tube in freezing water for another 5 minutes. Tissue precipitate will settle at the bottom of the test tube. d. Dilute the clear liquid, which is the DNA solution, 50 times to obtain the appropriate amount of DNA for the reaction. 1.1 Aspirate 10 microliters of the clear fraction and mix it with 490 microliters of distilled water. 1.2 Add 2 microliters of the clear fraction of the DNA solution extracted from the fish sample to the LAMP solution containing: a. 0.2 micromolar of Si-F3 outer primer (5’-TAATGGTGGCGGACACTT-3’), b. 0.2 micromolar of Si-B3 outer primer (5’-AAGTAATTTCAAGACTTCCTTCT-3’), and c. 0.2 micromolar of Si-FIP inner primer (5’-TGCACTTGCAACTTCTTTTGTTACTTTTTAATCATGCTTTGTTCTGGGA-3’). 2.0 microlars d. Inner primer Si-BIP, base sequence (5’-AAGAACAATTTGCAGCAGCTTTTAACAACTAACCAAGCCCA –3’) 2.0 microlars e. Loop primer Si-LF, base sequence (5’-ACTGGCCGTTTTGGTTCTGGT-3’) 2.0 microlars f. Loop primer Si-LB, base sequence (5’-GACCAACTTTTGGATC-3’) 2.0 microlars g. Buffer at 1x concentration (Triton-HCl 20 mmol, (NH4)2SO4 10 mmol, KCl 10 mmol, MgSO4 2 mmol, Triton X-100 0.1 percent, pH 8.8) h. Magnesium sulfate solution 4.0 mmol i. 1.2 Betaine 0.5 M, dNTPs 1.4 mmol, Bst enzyme DNA polymerase 8 units, Hydroxynaptal blue solution 120 micromol. 1.3 Heat all components to 63°C for 30 minutes. 1.4 Read the results visually immediately. In samples infected with Streptococcus iniae bacteria, the reaction solution will change from purple (negative test result) to blue (positive test result).