Primer set, kit and method for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses

A technology for detecting kits and iridescent viruses, which is applied in the field of molecular biology, can solve problems that have not been reported, and achieve the effects of avoiding false positives, increasing copy number, and easy operation

Pending Publication Date: 2020-05-08
HANGZHOU ALLSHENG INSTR +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are few reports about the detection kit of shrimp iridescent virus, and there are only patent documents that adopt RAA technology to detect shrimp iridescent virus, that is: application publication number is CN107988428A invention patent application discloses a kind of shrimp iridescent virus (SIV) RAA Constant-temperature fluorescence detection methods and detection kits, and the use of RPA reaction system to detect shrimp iridescent virus is also less, and the use of gene editing technology has not been reported

Method used

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  • Primer set, kit and method for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses
  • Primer set, kit and method for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses
  • Primer set, kit and method for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Experimental materials

[0045] Sample 1: Shrimp seedlings obtained from the Hangzhou Shrimp Larvae Base (provided by Zhejiang Aquatic Technology Extension Station).

[0046] Positive quality control product: Pet28a-CMP plasmid, the nucleotide sequence is shown in SEQ ID NO.5.

[0047] 2. Extraction of Shrimp Iridovirus DNA

[0048] (1) Weigh 30 mg of the experimental sample (take the whole shrimp seedlings), add 500 μL of physiological saline, mix upside down, remove the liquid, and repeat this step once;

[0049] (2) Put the washed shrimp seedlings in a grinding tube, add 1 grinding bead (8 mm in diameter), add 100 μL of lysate, put it into a homogenizer, and homogenize at 6 m / s for 20 s;

[0050] (3) Add 100 μL of lysate and 20 μL of proteinase K, and incubate at 56°C for 1 hour;

[0051] (4) Add 200 μL of binding solution and incubate at 70°C for 10 min;

[0052] (5) Add 200 μL of absolute ethanol, mix it upside down, add it to the adsorption column, and cent...

Embodiment 2

[0102] Embodiment 2 primer specificity experiment

[0103] 1. Detection method

[0104] In order to detect the primer specificity of the kit provided in Example 1, the CRISPR-Cas12a protease isothermal amplification detection method provided in the third part of Example 1 was used to detect viruses WSSV (prawn white spot syndrome virus), IHHNV (infectious Subcutaneous and hematopoietic necrosis virus), EHP (Shrimp Enterococcus hepatica), SHIV (Shrimp Iridescent Virus) were detected, and the detection status of the reagents on SHIV virus and other common viruses in prawns was analyzed.

[0105] 2. Test results

[0106] The test results show that only the virus SHIV sample has an "S" type amplification curve, and the negative control (ultrapure water) and virus WSSV, IHHNV, EHP samples do not appear to be amplified (such as image 3 shown).

[0107] The above experimental results show that the CRISPR-Cas12a protease isothermal amplification detection kit provided in Part 3 of...

Embodiment 3

[0108] Embodiment 3 Sensitivity experiment

[0109] 1. Detection method

[0110]Extract the positive plasmid (T vector containing the target sequence fragment), quantify the positive plasmid by NanoDrop One, and dilute it to 10pg / μL, 1pg / μL, 100ag / μL, 10ag / μL, 1ag / μL, 0.1ag / μL. The above-mentioned CRISPR-Cas12a protease detection method was used to amplify and detect the diluted positive plasmids at various concentrations.

[0111] 2. Test results

[0112] Test results such as Figure 4 As shown, the curves from left to right are the amplification results of positive standards of 10pg / μL, 1pg / μL, 100ag / μL, 10ag / μL, 1ag / μL, and 0.1ag / μL, from which it can be seen that the present invention The detection sensitivity of the CRISPR-Cas12a kit can reach 0.1ag / μL, and the accuracy is better than that of ordinary PCR detection methods, indicating that the CRISPR-Cas12a protease detection kit and detection method of the present invention have high sensitivity for the diagnosis of...

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Abstract

The invention discloses a primer set, kit and method for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses. The primer set includes a pair of RPA primers, crRNA anda molecular probe including fluorescent groups and quenching groups, and nucleotide sequences are shown as SEQ ID NO. 1-4. The provided primer set and kit for performing isothermal detection on CRISPR-Cas12a protease of prawn iridescent viruses can specifically identify the specific sequence of a prawn iridescent virus CMP gene, so that extremely high specificity and sensitivity can be achieved,the minimum detection limit can reach 0.1 ag / [mu]L; the primer set and kit are very stable and can effectively avoid false positive results generated by non-specific amplification; and the primer setand kit are suitable for the early monitoring on the inapparent infection of the prawn iridescent viruses.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a CRISPR-Cas12a protease isothermal detection primer set, kit and detection method for shrimp iridescent virus. Background technique [0002] Iridescent virus (abbreviated as SHIV) belongs to the Iridoviridae family and is a cytoplasmic, double-stranded DNA virus with icosahedral symmetry. The virus has a wide range of hosts and can infect fish, amphibians and invertebrates. Infection with the virus will cause basophilic inclusion bodies and nuclear pyknosis to appear in the hematopoietic tissues, gill filaments, hepatopancreas, appendages and muscle blood cells of shrimp. The main symptoms are swimming, lying on the side, reddish body, liver Pancreatic atrophy and jejunal emptying. [0003] For the prevention and treatment of the disease, scholars at home and abroad generally believe that comprehensive prevention is a relatively effective method, that is, to detect th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2545/113C12Q2563/107
Inventor 黄俊张徐俞杨稳丁雪燕郑晓叶付媛媛骆志成樊伟东
Owner HANGZHOU ALLSHENG INSTR
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