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PCR detection primers and detection method of prawn infectious hypodermal and hematopoietic necrosis virus

A technology of hematopoietic tissue and necrotic virus, applied in the field of molecular biology technology detection, can solve the problems of insufficient accuracy and stability, prone to missed detection, mutant strains, etc., and achieve high accuracy, high accuracy, and high sensitivity Effect

Inactive Publication Date: 2018-12-18
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method has the advantage of high sensitivity, but when carrying out IHHNV virus detection with published primers in the prior art, accuracy and stability are not enough, and sometimes missed detection easily occurs, for example, the detection of some diseased samples is negative, but existing Primers are not conserved in all strains, resulting in missed detection of some strains with variations in this region

Method used

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  • PCR detection primers and detection method of prawn infectious hypodermal and hematopoietic necrosis virus
  • PCR detection primers and detection method of prawn infectious hypodermal and hematopoietic necrosis virus
  • PCR detection primers and detection method of prawn infectious hypodermal and hematopoietic necrosis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Sensitivity detection

[0025] The primers for PCR detection of shrimp infectious subcutaneous and hematopoietic necrosis virus consist of upstream primers and downstream primers, and their nucleotide sequences are:

[0026] Upstream primer: 5'-AATTTCTCCAAGCCTTCTCACC-3'

[0027] Downstream primer: 5'-TCTGGCAGCAAAGGTAACTCC-3'.

[0028] Direct PCR detection:

[0029] (1) Dilute the infectious hypodermic and hematopoietic necrosis virus (IHHNV) template to 8 different concentration gradients, followed by 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 、10 -11 copy / μl.

[0030] (2) Using the above primers to carry out PCR amplification reaction to obtain 8 PCR amplification products;

[0031] The reaction system of the PCR amplification reaction is 20 μL, including 10 μL of 2×Taq PCR Master Mix, 0.5 μL of upstream primer and downstream primer, ddH 2 O 8.5 μL, DNA template 0.5 μL.

[0032] The program of the PCR amplification reaction is: 3 min at ...

Embodiment 2

[0037] Embodiment 2 specific detection

[0038] In order to verify the specificity of the PCR reaction of the present invention, a negative control experiment using double distilled water as a template is carried out. The reaction system is the same as in Example 1, except that the template uses ddH 2 O instead, after the completion of the PCR reaction, a second PCR reaction was carried out using the PCR reaction product as a template to eliminate factors such as artificial contamination in the laboratory. The results of electrophoresis of the product of the secondary reaction are shown in image 3 , wherein M is DNAMarkerII, swimming lanes 1-9 are negative controls, and swimming lanes 10 and 11 are positive controls.

Embodiment 3

[0040] During the period from April 23 to April 29, 2015, 144 female Macrobrachium rosenbergii female broodstock were subjected to secondary PCR detection three times respectively. The detection method:

[0041] (1) extract the genomic DNA of the sample to be tested;

[0042] (2) using the primers of Example 1 to carry out PCR amplification reaction on the genomic DNA of the sample to be tested, to obtain PCR amplification products;

[0043] The reaction system of the PCR amplification reaction is 20 μL, including 10 μL of 2×Taq PCR Master Mix, 0.5 μL of upstream primer and downstream primer, ddH 2 O 8.5 μL, DNA template 0.5 μL.

[0044] The program of the PCR amplification reaction is: 3 min at 94°C; 1 min at 94°C, 1 min at 68°C, 1 min at 72°C, 35 cycles; 10 min at 72°C.

[0045] (3) carry out agarose gel electrophoresis (concentration of agarose is 1.5%, voltage is 70V) with the PCR amplified product of step (2), judge whether to contain prawn infectious subcutaneous and h...

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Abstract

The invention relates to PCR detection primers of prawn infectious hypodermal and hematopoietic necrosis virus. The primers have the nucleotide sequences: 5'-AATTTCTCCAAGCCTTCTCACC-3' of an upstream primer and 5'-TCTGGCAGCAAAGGTAACTCC-3' of a downstream primer. Compared with primers in the prior art, the PCR detection primers of the prawn infectious hypodermal and hematopoietic necrosis virus havethe advantage of a wider measurement range, during primer designing, an IHHNV sequence, obtained from a decapoda crustacean, published in GenBank is referred to, variation sites of IHHNV among different groups are avoided, the part which is conservative in published sequences separately is selected for primer designing, the measured range is wider and stabler, and a result is more accurate and reliable. The PCR detection primers of the prawn infectious hypodermal and hematopoietic necrosis virus can be applied for IHHNV pathogen detection and early warning in domestic prawn culture and seedling, and have good prospects.

Description

technical field [0001] The invention belongs to the field of molecular biological technology detection, and in particular relates to a PCR detection primer and a detection method for infectious subcutaneous and hematopoietic tissue necrosis virus of prawns. Background technique [0002] Infectious hypodermic and hematopoietic necrosis virus (IHHNV) is a small single-stranded DNA virus. It is the smallest particle among known shrimp viruses. , Icosahedron (Icosahedron) symmetrical spherical particles. The parts of IHHNV infection in shrimp include ectodermal tissues such as gills, epidermis, front and rear intestinal epithelial cells, nerve cords and ganglions; and mesoderm organs such as hematopoietic tissues, antennal glands, Gonads, lymphoid organs, connective tissue and striated muscle. Inclusion bodies form in the host cell nucleus. In the white prawn (Litopenaeus vannamei), it causes Runt-deformity syndrome (RDS), which grows slowly and unevenly, the frontal angle is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2565/125
Inventor 李云刘红唐芳温贝妮朱其建戴习林
Owner SHANGHAI OCEAN UNIV
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