TH64843B - Sequence diagnosis for shrimp pathogens - Google Patents

Abstract

DC60 (15/01/51) An isolated diagnostic primer for Vibrio harvei detection. The primer is based on the LuxR gene region of Vibrio harvei and may be used in primer amplification or nucleic acid hybridization analysis methods. An isolated diagnostic primer for Vibrio harvei detection. The primer is based on the LuxR gene region of Vibrio harvei and may be used in primer amplification or nucleic acid hybridization analysis methods.

Claims (19)

1. A pair of primer sequences for the diagnosis of Vibrio harvey as mentioned in SEQ ID NO: 1 and SEQ ID NO: 2. 2. A test kit for the detection of Vibrio harvei consisting of a pair of primer sequences for the diagnosis of Vibrio harvei of claim 1. 3. An assay kit for the detection of Vibrio harvei pursuant to claim paragraph 2, which shall include at least one reagent selected from a group containing heat-resistant polymerase, a mixture of four different deoxynucleotide triphosphates, a nucleic acid-binding fluorescent molecule, at least one pair of internal sample control primers, at least one internal template control and at least one pair of internal template control primers, and a probe containing a sequence complementary to at least one region of nucleic acid within the Vibrio harvei genome which can be amplified with a pair of diagnostic Vibrio harvei primer sequences. 4. Methods for detecting the presence of Vibrio harvei in samples containing: (i) DNA preparation from samples suspected of containing Vibrio harvei; and (ii) DNA probing with probes obtained from the diagnostic primer sequences for Vibrio harvei isolated as described in SEQ ID NO: 1 or SEQ ID NO: 2 under appropriate hybridization conditions; where the identification of hybridizable nucleic acid regions confirms the presence of Vibrio harvei. 5. Method for detecting the presence of Vibrio harvei in samples under claim 4, whereby probes obtained from isolated diagnostic primer sequences of Vibrio harvei are selected from groups containing SEQ ID NO:2, SEQ ID NO:3, and their complete complementary sequences. 6. Method pursuant to claim 4, where the probe has a replication inhibition moiter at the 3' end. 7. The method under claim 6, whereby the replication inhibition moitity is selected from groups consisting of a dideoxynucleotide, a 3' deoxynucleotide, an unpaired nucleoside or nucleotide sequence, a 3' phosphate group and a chemical. 8. Method according to claim 7, where the 3' deoxygenated nucleotide is cordycepin. 9. The method for detecting the presence of Vibrio harvei in samples shall be: (i) preparation of DNA from samples suspected to contain Vibrio harvei; and (ii) probe the DNA with probes obtained from the Vibrio harvei diagnostic primer sequences isolated as described in SEQ ID NO: 1 or SEQ ID NO: 2, in which the amplification product is generated; where the identification of hybridizable nucleic acid regions confirms the presence of Vibrio harvei. 10. Method for the detection of the presence of Vibrio harvei in samples according to claim No. 9, where the increase of (ii) is carried out by the use of polymerase chain reaction. 11. Method for the detection of the presence of Vibrio harvei in samples according to claim paragraph 9, where the amplification of (ii) is carried out in the presence of a fluorescent binder, nucleic acid or fluorescently labeled probe and the presence of the amplification product is confirmed by the use of fluorescence detection. 12. The procedure under claim 11, where fluorescence-labeled probes are selected from groups comprising SEQ ID NO:8 and SEQ ID NO:9. 13. The method pursuant to claim 9, where at least one pair of internal sample control primers are incorporated into the number of (ii) to produce the internal sample control product. 14. Procedure pursuant to claim 13, where at least one pair of internal sample control primers is selected from groups comprising SEQ ID NOs: 4 and 5, and SEQ ID NOs: 6 and 7. 15. The method pursuant to claim paragraph 9, where at least one pair of internal sample control primers and at least one internal template control are incorporated in increasing the number of (ii) to produce the internal sample control product. 16. Methods for the detection of Vibrio harvei in samples containing: (i) preparation of DNA from samples suspected of containing Vibrio harvei; (ii) DNA amplification with probes obtained from the Vibrio harvei diagnostic primer sequences mentioned in SEQ ID NO: 1 or SEQ ID NO: 2 by thermal cyclization at least the denaturation and amplification temperatures in the presence of a nucleic acid-binding fluorescent agent or fluorescently labeled probe; (iii) measurement of the amount of fluorescence generated by the nucleic acid-binding fluorescent agent or fluorescently labeled probe during thermal cyclization; (iv) measurement of the threshold cycle at which the amount of fluorescence generated by the nucleic acid-binding fluorescent agent or fluorescently labeled probe reaches a threshold value higher than the baseline; and (v) calculation of the amount of Vibrio. The Vibrio harvei cytokinesis in the sample was analyzed by comparing the number of threshold cycles measured for Vibrio harvei in the sample with the standard curve of the number of threshold cycles per logarithm of the template concentration measured by measuring a standard solution of known concentration. 17. The procedure under claim 16, where fluorescence-labeled probes are selected from groups comprising SEQ ID NO:8 and SEQ ID NO:9. 18. Any of the methods under Claim 4, 9, or 16, where the method is used to assess the non-stimulation of Vibrio harvei. 19. Any of the methods under Claims 4, 9, or 16, where methods are used in combination, are chemical treatments to improve the health and growth of shrimp.