A primer for inhibiting specific amplification of shrimp and oyster 18srDNA sequences and its application

A specific, oyster technology, applied in the field of PCR primers, to achieve the effect of convenient research

Active Publication Date: 2022-05-27
广州鸿真生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no research report on the primers used to inhibit the specific amplification of shrimp and oyster 18S rDNA sequences in the prior art

Method used

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  • A primer for inhibiting specific amplification of shrimp and oyster 18srDNA sequences and its application
  • A primer for inhibiting specific amplification of shrimp and oyster 18srDNA sequences and its application
  • A primer for inhibiting specific amplification of shrimp and oyster 18srDNA sequences and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Design of Suppression Primers

[0024] Design scheme of suppressor primers:

[0025] 1. Design of the C3 spacer

[0026] The 5' end (18-25nt) has a certain overlap with the 3' end of F(R) of the universal primer, and the subsequent sequence should be as consistent as possible with the 18S gene sequence to be suppressed, which is different from the gene sequence that does not want to be suppressed, and finally at the end Plus C3spacer.

[0027] 2. Design of Dual Prime Oligonucleotides

[0028] The 5' end is similar in design to the 5' end of C3, except that 44444 (4=dlnosine) is added before the 3' end (6-12nt), and a C3 spacer is added at the end.

[0029] Suppression primer design process:

[0030] like figure 1As shown, according to Penaeus vannamei (GenBank accession no. EU920969.1), Penaeus vannamei (GenBank accession no. EU118282.1), Penaeus vannamei (GenBank accession no. AF186250.1), AF124597.1), Penaeus vannamei (GenBank accession no. AF463509.1)...

Embodiment 2

[0035] Example 2 Verification of Inhibition Primers

[0036] The DNA of Penaeus vannamei and Hong Kong oyster were extracted using a mollusk DNA extraction kit (Magen, Suzhou, China). ) The specific operation is carried out according to the instructions in the manual.

[0037] For the inhibitory primer nmb-BP2-DPO, different concentrations of 0.01μM, 0.02μM, and 0.04μM were used to configure the inhibition reaction system. 2 O was used as a negative control, and the reaction without inhibitory primers was used as a positive control. The templates were respectively the DNA of the digestive tract of Penaeus vannamei, the DNA of the digestive tract of Hong Kong oyster, the DNA of planktonic microorganisms in the water body filtered on the membrane, and the DNA of zooplankton. Three replicates of different template DNAs were set up and amplified by a PCR machine.

[0038] Among them, the 18S universal primer sequence used is:

[0039] 1391F: GTACACACCGCCCGTC

[0040] EukBr:TGA...

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PUM

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Abstract

The invention discloses a primer for inhibiting the specific amplification of shrimp and oyster 18S rDNA sequences. The primer mainly consists of the base sequence shown in SEQ ID NO.1, between the base sequences AG It consists of five inserted deoxyhypoxanthines, and a C3 spacer at the end of the base sequence. The primer can effectively inhibit the amplification of host genes and improve the amplification effect of 18S rDNA of other eukaryotic organisms (such as protozoa, algae, fungi) in the host. The invention also discloses the application of the above-mentioned primers in the preparation of reagents capable of inhibiting the specific amplification of shrimp and oyster 18S rDNA sequences.

Description

technical field [0001] The invention belongs to the technical field of PCR primers, in particular to a primer for inhibiting the specific amplification of shrimp and oyster 18S rDNA sequences and its application. Background technique [0002] The genomes of eukaryotic hosts (eg, oysters, shrimp) contain 18S rDNA gene sequences. At the same time, its body (such as the intestine) also carries a variety of eukaryotic microorganisms (such as undigested algal food, endosymbiotic fungi, parasites), and their genomes also contain 18S gene sequences, and the 18S sequences among different species are certain similarity. [0003] In order to study the eukaryotic microorganisms existing in the host, people usually use PCR method to carry out specific amplification through the universal primer of 18S gene. However, due to the unavoidable interference of a large number of host's own 18S gene sequences, the host 18S sequences in the amplified products account for most of the proportion,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/10C12N15/113
Inventor 姜敬哲戚认杰刘聪翟立广
Owner 广州鸿真生物科技有限公司
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