Specific primers, probes and fast detection kit for macrobrachium rosenbergii ronivirus-1

A primer-probe and kit technology, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of immeasurable economic losses in the macroprawn industry, improve detection sensitivity, prevent Pollution, easy operation effect

Active Publication Date: 2020-03-10
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus has caused incalculable economic losses to the Macrobrachium industry in recent years

Method used

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  • Specific primers, probes and fast detection kit for macrobrachium rosenbergii ronivirus-1
  • Specific primers, probes and fast detection kit for macrobrachium rosenbergii ronivirus-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Primer-probe mixture for detecting Macrobrachium prawn hood virus-1, which consists of primer set A and Taqman fluorescent probe B, the 5' end of probe B is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescence quencher group.

[0027] The primer set A comprises primer MrRoV-q119F and primer MrRoV-q119R;

[0028] The nucleotide sequence of the primer MrRoV-q119F is shown in SEQ ID NO: 1;

[0029] The nucleotide sequence of the primer MrRoV-q119R is shown in SEQ ID NO: 2;

[0030] The nucleotide sequence of B of the probe is shown in SEQ ID NO:3.

[0031] Wherein, the fluorescent reporter group is selected from 6-carboxyfluorescein (6-FAM), hexachloro-6-methylfluorescein (HEX), VIC fluorescent dye (VIC), tetrachloro-6-carboxyfluorescein (TET) , Carboxy-X-rhodamine (ROX), 6-carboxytetramethylrhodamine (TAMRA), sulfonyl rhodamine (Texas Red), 6-carboxy-4', 5'-dichloro-2', 7' - one of dimethoxyfluorescein succinimidyl ester (JOE), cyan...

Embodiment 2

[0037] Macrobrachium rod sleeve virus-1 fluorescent quantitative detection kit consists of individually packaged RT-qPCR reaction solution, positive quality control, negative quality control and primer-probe mixture.

[0038] The primer-probe mixture is composed of the primer set A of Example 1 and the Taqman fluorescent probe B, wherein the final concentration of the upstream and downstream primers of the primer set A is 0.2 μM, and the final concentration of the probe is 0.1 μM.

[0039] Taqman fluorescent probe B, its nucleotide sequence is marked with 6-FAM at the 5' end and BHQ1 at the 3' end;

[0040] The negative quality control product is sterilized normal saline; the positive quality control product is based on the purified Macrobrachium prawn sleeve virus-1 genome as a template, PCR amplification is carried out by primer set A, the amplified product is connected to the carrier, and the correctness is confirmed by gene sequencing. Pseudovirus obtained from packaging; ...

Embodiment 3

[0043] Macrobrachium rod sleeve virus-1 fluorescent quantitative detection kit was applied, and the sample to be tested was extracted with a commercial RNA extraction kit for total RNA, and 3uL RNA and 3uL of the primer-probe mixture of Example 1 were added to the reaction tube, and then added 19uLRT-qPCR reaction solution, after mixing, perform one-step fluorescence quantitative RT-qPCR, the reaction conditions are: 42°C for 20 minutes, 95°C for 5 minutes, 95°C for 10 seconds, 60°C for 40 seconds, a total of 40 cycles; Set it to FAM when collecting signals, and end the reaction at 60°C;

[0044] Result judgment:

[0045] If there is an S-type amplification curve in the detection channel, and the Ct value is ≤ 35, then Macrobrachium rod virus-1 is judged as positive; if there is no S-type amplification curve in the detection channel, Macrobrachium rod virus-1 is judged as negative; S-type amplification curve appears in the channel, and when the Ct value is >35, it needs to be...

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Abstract

The invention discloses a primer and probe mixed liquid for detecting macrobrachium rosenbergii ronivirus-1. The mixed liquid is composed of a primer group A and a Taqman fluorescent probe B. The end5' of the Taqman fluorescent probe B is marked with a fluorescent reporter group, and the end 3' of the Taqman fluorescent probe B is marked with a fluorescent quenching group. The primer group A comprises a primer MrRoV-q119F and a primer MrRoV-q119R, the nucleotide sequence of the primer MrRoV-q119F is shown in the formula of SEQ ID NO: 1, the nucleotide sequence of the primer MrRoV-q119R is shown in the formula of SEQ ID NO: 2, and the nucleotide sequence of the Taqman fluorescent probe B is shown in the formula of SEQ ID NO: 3. The invention discloses a fluorescent quantitative detection kit for macrobrachium rosenbergii ronivirus-1. The kit comprises the mixed liquid, an RT-qPCR reaction solution, a positive quality control material and a negative quality control material, the RT-qPCRreaction solution is a probe-method fluorescent quantitative one-step RT-qPCR reaction solution, the negative quality control material is sterilized normal saline, and the positive quality control material is a carrier containing macrobrachium rosenbergii ronivirus-1 replicase genes. The kit is used for detecting the macrobrachium rosenbergii ronivirus-1 and is high in speed and high in specificity.

Description

technical field [0001] The invention belongs to the field of rapid detection of target RNA fragments, and in particular relates to a specific primer, a probe and a rapid detection kit for detecting Macrobrachium bark virus-1. Background technique [0002] Macrobrachium rosenbergii Ronivirus-1 (Macrobrachium rosenbergii Ronivirus, MrRoV) is a pathogenic virus that causes a large number of deaths in the larvae and culture stages of Macrobrachium prawns. A large number of acute deaths occur in the process of shrimp, and it can also cause damage to the gills of adult shrimp during the breeding period and cause death, which is extremely harmful to the macrobrachium nursery industry and aquaculture industry. MrRoV is a single-stranded RNA virus belonging to the family Roniviridae of the order Nidovirales. Today, there is only one genus Okavirus under this family, and there is only one species under this genus, namely the yellow-headed shrimp Virus (Yellow head virus). Macrobrach...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2561/101C12Q2563/107C12Q2521/107Y02A40/81
Inventor 潘晓艺沈锦玉蔺凌云姚嘉赟尹文林曹铮
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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