Primer pairs, method and fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses
A technique for red-spotted grouper and nerve necrosis, which is applied in the field of biological identification of aquatic biological diseases, and achieves the effects of mild reaction conditions, reduced background influence and improved specificity
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Embodiment 1
[0064] Example 1 Primer set for detection of red grouper neuronecrosis virus and effectiveness detection
[0065] In this example, according to the published genome sequence of red-spotted grouper neuronecrosis virus, the specific sequence of red-spotted grouper neuronecrosis virus gene is selected, and then analyzed and designed to be used in LAMP detection technology, which can specifically identify red The specific primer group of grouper neuronecrosis virus, this primer group is made up of following four primers:
[0066] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 1;
[0067] Internal primer BIP, the nucleotide sequence of which is shown in SEQ ID NO: 2;
[0068] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 3;
[0069] The nucleotide sequence of the outer primer B3 is shown in SEQ ID NO:4.
[0070] The present embodiment selects 8 positive fishes and 2 healthy fishes detected by RT-PCR (such as figure 1 show...
Embodiment 2
[0072] The preparation of embodiment 2 rapid diagnostic kits
[0073] The rapid diagnostic kit of this embodiment can be used for the rapid diagnosis of whether red-spotted grouper neuronecrosis virus is contained in the sample by the loop-mediated isothermal amplification technique. Composition of reaction solution, Bst DNA polymerase, positive control substance, chromogenic solution and foam plate, wherein:
[0074] (1) Primer set for detection of red grouper neuronecrosis virus: DNA synthesizer, 1 tube, inner primer F3, outer primer B3, inner primer FIP and inner primer BIP, inner primer FIP, its nucleotide sequence As shown in SEQ ID NO: 1; inner primer BIP, its nucleotide sequence is shown in SEQ ID NO: 2; outer primer F3, its nucleotide sequence is shown in SEQ ID NO: 3; outer primer B3, its The nucleotide sequence is shown as SEQ ID NO: 4, and the concentrations of the above four primers are 10uM respectively;
[0075] (2) Configure LAMP reaction solution: containing ...
Embodiment 3
[0089] The preparation of embodiment 3 rapid diagnostic kits
[0090]The rapid diagnostic kit of this embodiment can be used for the rapid diagnosis of whether red-spotted grouper neuronecrosis virus is contained in the sample by the loop-mediated isothermal amplification technique. Reaction solution, Bst DNA polymerase, positive control substance, chromogenic solution, Trizol, chloroform, isopropanol, 70% ethanol, RNase-free water, RT reaction solution, reverse transcriptase and foam plate, in which:
[0091] (1) Primer set for detection of red grouper neuronecrosis virus: DNA synthesizer, 1 tube, inner primer F3, outer primer B3, inner primer FIP and inner primer BIP, inner primer FIP, its nucleotide sequence As shown in SEQ ID NO: 1; inner primer BIP, its nucleotide sequence is shown in SEQ ID NO: 2; outer primer F3, its nucleotide sequence is shown in SEQ ID NO: 3; outer primer B3, its The nucleotide sequence is shown as SEQ ID NO: 4, and the concentrations of the above f...
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