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Procypris rabaudi real-time fluorescent PCR (polymerase chain reaction) amplification primer, probe and detection method based on environmental DNA (deoxyribonucleic acid)

A technology of real-time fluorescence and amplification primers, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the difficulty in designing fluorescent-specific Taqman probes, the limited biological information of Iwahara carp conservation, and the distribution The scope and population situation are not clear, and the results are superior to human-intuitive judgment results, shortened detection time, and accurate monitoring results

Active Publication Date: 2021-11-19
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limited by the current observation and sampling techniques, the conservation biology information about Iwahara carp is quite limited, and its distribution range and population are still unclear
Moreover, there are many kinds of fishes in the Yangtze River, and the differences at the molecular level of species with close geographical distribution are also small, which causes certain difficulties in the design of real-time fluorescence-specific Taqman probes, which is a technical problem in the field

Method used

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  • Procypris rabaudi real-time fluorescent PCR (polymerase chain reaction) amplification primer, probe and detection method based on environmental DNA (deoxyribonucleic acid)
  • Procypris rabaudi real-time fluorescent PCR (polymerase chain reaction) amplification primer, probe and detection method based on environmental DNA (deoxyribonucleic acid)
  • Procypris rabaudi real-time fluorescent PCR (polymerase chain reaction) amplification primer, probe and detection method based on environmental DNA (deoxyribonucleic acid)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The real-time fluorescent PCR detection method of environmental DNA of carp iwahara comprises the following steps:

[0025] 1. Design primers and probes.

[0026] The following primers YYLF1 / YYLR1 and probe YYL-MGB1 were designed according to the 12SrRNA gene sequence in the mitochondrial DNA (mtDNA) of carp Iwahara (primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.).

[0027] The sequence of this primer is: upstream primer YYLF1 nucleotide sequence as shown in SEQ ID No.1; Downstream primer YYLR1: nucleotide sequence as shown in SEQ ID No.2; Probe YYL-MGB1: nucleotide sequence as shown in As shown in SEQ ID No.3, the fluorescent group is connected to the 5' end of the probe, and the quenching group is connected to the 3' end.

[0028] The detection principle of the above primers and probes is as follows: figure 1 shown. Probe YYL-MGB1 is an oligonucleotide probe with a fluorophore attached to the 5' end of the probe and a quencher at ...

Embodiment 2

[0035] Iwahara carp primer YYLF1 / YYLR1 and probe YYL-MGB1 specificity verification and sensitivity test, including the following steps:

[0036] 1. Extract DNA.

[0037] Take Iwahara carp and other fish muscles (see Table 1 for samples and sources) and put them into a mortar, add liquid nitrogen to grind them into powder, and use DNeasy Blood & Tissue Kit (Qiagen Company) to extract sample DNA.

[0038] Table 1 Specific information of tested samples

[0039] serial number serial number species name place of origin Quantity (bar) 1 YYL Iwahara carp Yibin City, Sichuan Province 1 2 JY crucian carp Beibei District, Chongqing 1 3 HSY Yellow catfish Beibei District, Chongqing 1 4 N Q loach Beibei District, Chongqing 1 5 HS eel Beibei District, Chongqing 1 6 LY Silver carp Beibei District, Chongqing 1 7 YY bighead carp Beibei District, Chongqing 1 8 BDCWH channel catfish Beibei D...

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Abstract

The invention discloses a composition of a primer and a probe for procypris rabaudi real-time fluorescent PCR (Polymerase Chain Reaction) detection based on environmental DNA (Deoxyribose Nucleic Acid), and establishes a real-time fluorescent PCR detection method of procypris rabaudi to achieve the purpose of accurately identifying procypris rabaudi. A specific primer is designed according to the gene sequence of 12SrRNA in mitochondrial DNA of procypris rabaudi, the nucleotide sequence of an upstream primer YYLF1 is as shown in SEQ ID No. 1, the nucleotide sequence of a downstream primer YYLR1 is as shown in SEQ ID No. 2, and a probe YYL-MGB1 has the nucleotide sequence as shown in SEQ ID No. 3. The probe can be applied to qualitative detection of procypris rabaudi in a water body only by collecting a water sample of a water area in which the procypris rabaudi live, and is high in specificity and good in timeliness; and the density of the procypris rabaudi in the environment can be quantitatively detected, and the biomass and time-space dynamics of the procypris rabaudi at different points can be reflected, so that a reference is provided for artificial release effect evaluation and resource management protection of the procypris rabaudi under the large background of procypris rabaudi without fishery in the Yangtze River.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an environmental DNA-based real-time fluorescent PCR amplification primer and probe for carps and a detection method thereof. Background technique [0002] Yanyuan carp (Procypris rabaudi Tchang) belongs to Cypriniformes (Cypriniformes), Cyprinidae (Cyprinidae), Procypris (Procypris), is a unique fish in the upper reaches of the Yangtze River and its tributaries, mainly distributed in the stems of the Yangtze River system above Yichang. In the rivers, tributaries and the middle and lower reaches of the Jinsha River, it belongs to the first-class edible precious fish. The wild resource of Iwahara carp has declined sharply since the 1970s, and it is listed as a vulnerable species and an endangered fish. Limited by the current observation and sampling techniques, up to now, the conservation biological information about Iwahara carp is quite limited, and its distribution range and popul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2563/107C12Q2561/113Y02A40/81
Inventor 何文平闫卉果董智玲张连博马婷婷
Owner SOUTHWEST UNIVERSITY
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