A group of primers and detection method for detecting iridovirus
A technology of an iridescent virus and a detection method, which is applied in the field of rapid detection of iridescent virus, can solve the problems of short reaction time, long time, limited use range, etc., and achieves the effects of short reaction time, simple operation and strong specificity.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0030] A set of primers for detecting iridovirus, said primers are complementary to a part of the nucleotide sequence of the iridovirus or its complementary chain, and can specifically amplify the nucleotide sequence of the iridovirus under the action of DNA polymerase a part of. The primers are oligonucleotides shown in SEQ ID NO.1-6.
[0031] Table 1 Primer Sequence
[0032]
[0033] The above primers of SEQ ID NO.1-6 correspond to the primers of SEQ ID NO.1-6.
[0034] The primer for detecting the iridescent virus of the invention has high sensitivity and strong specificity. The 6 primers recognize 8 regions in total, fully guaranteeing their specificity. Among them, F3 and B3 are outer primers, which work when the amplification reaction is initiated; FIP and BIP are called inner primers, which work throughout the entire amplification process, and are the most critical pair of primers, resulting in a circular stem-loop The structure can be infinitely amplified; FL an...
Embodiment 2
[0036] A method for detecting iridovirus, the method is used to detect whether there is iridovirus in a sample, and the method adopts the primer set used for detecting iridovirus of the present invention, through loop-mediated isothermal amplification method (LAMP), specificity A part of the nucleotide sequence of the iridovirus is amplified accurately, and the presence or absence of the amplified product is confirmed.
[0037] LAMP amplification mainly utilizes two characteristics of large fragments of BstDNA polymerase: one is that DNA polymerase has 5'→3' DNA polymerase activity, and can amplify DNA nucleic acid at a constant temperature; the other is that BstDNA polymerase does not have 5 '→3' exonuclease activity, which can displace (replace) the newly synthesized nucleic acid single strand from the newly generated DNA double strand, forming two independent nucleic acid single strands to start the next round of DNA nucleic acid synthesis; and The step of DNA denaturation ...
Embodiment 3
[0060] A method for detecting iridovirus, said method comprising the steps of:
[0061] (1) Prepare test samples: fresh or cell cultures stored at -80°C to 4°C, which can be directly used for total DNA extraction after thawing; fresh or cell cultures stored at -80°C to 4°C with phosphate buffer Seawater fish tissue disease material, fully homogenized, centrifuged at 10,000rpm for 10-30min, and the supernatant was used for the extraction of total DNA;
[0062] (2) Extract the total DNA of the sample: use D extraction reagent (product of Dalian Bao Biological Engineering Co., Ltd.), and operate according to the instructions of the reagent;
[0063] (3) Perform loop-mediated isothermal nucleic acid amplification reaction on the DNA in step (2), using Bst large-fragment DNA polymerase (product of New England Company), the reaction temperature is 60-65°C, and the reaction time is 10-60 minute( figure 2 );
[0064] (4) Terminate the reaction; 80°C for 5 minutes;
[0065] (5) De...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More 
PatSnap Eureka turns technology decisions into work you can execute. Powered by our Innovation Knowledge Graph, it runs expert workflows across engineering, life sciences, materials and intellectual property. Get your review-ready output in minutes.
