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RPA primer, probe and kit for detecting shell parasitic perkinsus

A technology of sending Qin worms and a kit, which is applied in the field of rapid diagnosis of aquatic animal parasites, can solve problems such as ineffective drug treatment and inability to guarantee drug concentration, and achieve the effects of visualization of test results, great potential for promotion and application, and simple operation.

Pending Publication Date: 2019-06-21
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As we all know, shellfish are mainly cultured in open seas. Once disease occurs, the drug concentration cannot be guaranteed like closed culture (such as pond culture), so drug treatment is difficult to be effective

Method used

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  • RPA primer, probe and kit for detecting shell parasitic perkinsus
  • RPA primer, probe and kit for detecting shell parasitic perkinsus
  • RPA primer, probe and kit for detecting shell parasitic perkinsus

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 Primer and probe design

[0038] Primer design: Using the ribosomal DNA internal transcriptional spacer sequences of the seven Piedia species discovered so far as design templates, a pair of primers that can amplify all Piedias were designed and screened. The primer sequences are:

[0039] Upstream primer F1: 5'-CGATGAAGGACGCAACGAAGTG-3' (SEQ ID NO.1);

[0040] Downstream primer R1: 5'-CAAGCGGGATACAAAGCATTAGATT-3' (SEQ ID NO.2).

[0041] A probe is designed in the amplified target sequence, and the probe Probe sequence is: 5'-CAGAATTCCGTGAACCAGTAGAAAATCTCAACGCATACTGCACAAAGGGGA-3' (SEQ ID NO.3).

[0042] In order to directly detect the results with test strips, a biotin label was added to the 5' end of the primer R1, a FAM label was added to the 5' end of the probe, dSpacer or THF was added in the middle, and a C3Spacer label was added to the 3' end.

[0043] The sequence after fluorescent labeling is as follows:

[0044] R1': 5'-biotin-CAAGCGGGATACAAAGCA...

Embodiment 2

[0049] The present embodiment provides the method that adopts RPA method to detect shellfish parasitic Pythias, comprising the following steps:

[0050] (1) Refer to Example 1 for primer design.

[0051] (2) DNA extraction: DNA from gill tissues of different shellfish is extracted for the detection of Piedia worms.

[0052] (3) Constant temperature amplification of recombinase polymerase: use the DNA of shellfish gill tissue as a template, use F1, R1' as upstream and downstream primers, and Probe' as probe to carry out constant temperature amplification. The amplification system is 50 μL, and the Rehydration 29.5 μL of buffer, 2.1 μL of upstream and downstream primers (10 μM), 0.6 μL of probe, 12.2 μL of sterilized ultrapure water, and 1 μL of DNA sample, a total of 47.5 μL of the system was pre-mixed, transferred to a reaction tube, and then 2.5 Add μL of MgAc (280mM) solution to the corresponding tube cap, mix well, and let it react at room temperature for 20-25min.

[0053]...

Embodiment 3

[0055] Sensitivity experiment of the diagnostic method of the present invention

[0056] (1) Amplify the ITS of Perkinsus beihaiensis using the general primers Perkinsus beihaiensis recommended by OIE Sequence, prepare the recombinant plasmid of this target sequence, and extract the plasmid DNA of the North Sea Piedia worm.

[0057] (2) Dilute the DNA concentration (copies / μL) of the North Sea Piedia worm plasmid to 2.6×10 5 , 2.6×10 4 , 2.6×10 3 , 2.6×10 2 , 2.6×10 1 , 2.6×5, 2.6×1, as DNA standards for sensitivity analysis.

[0058] (3) Using different concentrations of Beihai Paiqinia plasmid DNA as a template, using F1, R1' as upstream and downstream primers, and Probe' as a probe for constant temperature amplification, the amplification system is 50 μL, first rehydration buffer 29.5 μL, upper 2.1 μL of each downstream primer (10 μM), 0.6 μL of probe, 12.2 μL of sterilized ultrapure water, and 1 μL of DNA sample, a total of 47.5 μL of the system was pre-mixed, transf...

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Abstract

The invention discloses an RPA primer and a probe for detecting shell parasitic perkinsus. Sequences of the primer are shown as SEQ ID NO.1 and SEQ ID NO.2, a sequence of the probe is shown as SEQ IDNO.3, a biotin marker is added to 5' terminal of a downstream primer R1, an FAM marker is added to 5' terminal of the probe, a C3Spacer marker is added to 3' terminal, and dSpacer or THF is added intothe probe for modification. The invention further discloses a kit comprising the primer and the probe, application of the primer and the probe in detecting shell parasitic perkinsus and a method of adopting an RPA process to detect shell parasitic perkinsus. The primer, the probe, the kit and the method are short in detection time, high in sensitivity and specificity, low in requirement on operation environment and wide in application range.

Description

technical field [0001] The invention belongs to the technical field of rapid diagnosis of aquatic animal parasites, and in particular relates to an RPA primer, a probe and a kit for detecting shellfish parasite Pythias. Background technique [0002] Perkinsus spp. is a pathogenic parasite that can parasitize a variety of seawater shellfish, and has a wide distribution range. Slow growth and even lead to mass die-off of shellfish. The World Organization for Animal Health (OIE) stipulates that two of the most harmful species of pike worms (P. marinus and P. olseni) are pathogenic parasites that must be detected and reported during outbreaks of aquatic shellfish diseases. [0003] The life cycle of Piedia worms is simple, and its transmission does not require an intermediate host. Shellfish can complete the mutual infection and transmission of Piedias worms through behaviors such as feeding and excretion. Pieces are highly infective and can infect shellfish at all stages of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6844
Inventor 叶灵通吴霖王兆瑞王江勇
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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