Specific primer, probe and quick detection kit for detecting macrobrachium rosenbergii flavivirus-1

A flavivirus and kit technology, applied in the field of rapid detection of target RNA fragments, can solve problems such as immeasurable economic losses, and achieve the effects of omitting electrophoresis steps, preventing contamination, and improving detection sensitivity

Active Publication Date: 2020-02-28
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viral iron shrimp disease has occurred in major giant macrobrachium rosenbergii farms in my country for many years, and can be transmitted vertically through virus-infected species of shrimp, which has caused immeasurable economic losses to my country's macrobrachium rosenbergii nursery and aquaculture industry

Method used

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  • Specific primer, probe and quick detection kit for detecting macrobrachium rosenbergii flavivirus-1
  • Specific primer, probe and quick detection kit for detecting macrobrachium rosenbergii flavivirus-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Primer-probe mixture for detecting Macrobrachium flavivirus-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 fluorescent quencher group. The primer set A comprises primer MrFV-q150F and primer MrFV-q150R; the nucleotide sequence of the primer MrFV-q150F is shown in SEQ ID NO: 1; the nucleotide sequence of the primer MrFV-q150R is as shown in SEQ ID NO : shown in 2; the nucleotide sequence of the probe B is shown in SEQ ID NO: 3. 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), sulfonylrhodamine (Texas Red), 6-carboxy-4', 5'-dichloro-2', 7'-Dimethoxyfluorescein succinimidyl ester (JOE), cyanine 3 (Cy3), cyanine 3.5 (Cy3.5), cyanine 5 (Cy5), cyanin...

Embodiment 2

[0034] Macrobrachium Flavivirus-1 Fluorescent Quantitative Detection Kit consists of individually packaged RT-qPCR reaction solution, positive quality control, negative quality control and primer-probe mixture.

[0035]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.

[0036] Taqman fluorescent probe B, its nucleotide sequence is marked with 6-FAM at the 5' end and BHQ1 at the 3' end;

[0037] The negative quality control product is sterilized normal saline; the positive quality control product is based on the purified Macrobrachium flavivirus-1 genome as a template, PCR amplification is carried out by primer set A, the amplified product is connected to the carrier, and it is packaged after being confirmed to be correct by gene sequencing The resulting pseudovirus;

[...

Embodiment 3

[0040] Macrobrachium flavivirus-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 fluorescent 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; The signal collection is set to FAM, the end of the reaction at 60°C;

[0041] Result judgment:

[0042] If there is an S-type amplification curve in the detection channel, and the Ct value is ≤ 35, the macrobrach flavivirus-1 is judged as positive; if there is no S-type amplification curve in the detection channel, the macrobrach flavivirus-1 is judged as negative; When an S-type amplification curve appears and the Ct value is >35, re-testing is required. If the Ct ...

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Abstract

A primer probe mixed solution for detecting macrobrachium rosenbergii flavivirus-1 comprises a primer set A and a Taqman fluorescent probe B. The 5' end of the Taqman fluorescent probe B is marked with a fluorescent reporting group, and the 3' end of the Taqman fluorescent probe B is marked with a fluorescent quenching group. The primer set A comprises a primer MrFV-q150F and a primer MrFV-q150R.The nucleotide sequences of the primer MrFV-q150F and the primer MrFV-q150R are respectively represented by SEQ ID N0:1 and SEQ ID N0:2. The nucleotide sequence of the Taqman fluorescent probe B is represented by SEQ ID N0:3. A fluorescent quantitative detection kit for the macrobrachium rosenbergii flavivirus-1 comprises the mixed solution, an RT-qPCR reaction solution, a positive quality controland a negative quality control. The RT-qPCR reaction solution is an RT-qPCR reaction solution applied to the probe method and the fluorescent quantitation one-step method, the negative quality control is sterile normal saline, and the positive quality control is a vector containing polymerase genes of the macrobrachium rosenbergii flavivirus-1. The kit is used for detecting the macrobrachium rosenbergii flavivirus-1, and the speed and specificity are high.

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 flavivirus-1. Background technique [0002] Macrobrachium rosenbergii Flavivirus-1 (Macrobrachium rosenbergii Flavivirus-1, MrFV-1) is a viral pathogen that causes Macrobrachium rosenbergii to grow up, especially Macrobrachium rosenbergii, and often causes Macrobrachium rosenbergii "viral iron shrimp syndrome". The prematurity of the gonads of Macrobrachium rosenbergii during the breeding period caused huge losses to the production of Macrobrachium rosenbergii. The virus is also called vacuolization virus because it is easy to cause cytoplasmic vacuolization of specific cells of Macrobrachium rosenbergii. MrFV-1 is a single-stranded RNA virus that can infect larvae and adults of Macrobrachium rosenbergii. Macrobrachium rosenbergii "viral iron shrimp syndrome" first ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2563/107C12Q2561/101C12Q2531/113C12Q2547/101Y02A50/30
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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