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Gene marker for monitoring water pollution source in real time and application of gene marker

A genetic marker and real-time monitoring technology, applied in the field of bio-environmental technology detection, can solve the problems of being unable to locate water pollution sources and monitor water pollution in real time, and achieve excellent sensitivity effects

Pending Publication Date: 2020-07-28
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the biological indicators for water quality monitoring are the number of Escherichia coli and the total number of bacteria. However, these indicators can only reflect the status of the water body, and cannot monitor water body pollution in real time, nor can it locate the source of water body pollution

Method used

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  • Gene marker for monitoring water pollution source in real time and application of gene marker
  • Gene marker for monitoring water pollution source in real time and application of gene marker
  • Gene marker for monitoring water pollution source in real time and application of gene marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Primer Design

[0029] 224 animal feces samples (chicken, duck, human, dog, pig, cow) were collected, and 984,224 bacterial 16SrDNA sequences were obtained by high-throughput sequencing. By comparing the obtained sequences to the database, the 16SrDNA gene sequences of Faecalibacterium in all feces samples were selected, and a detailed comparison between species was performed, and a pair of DNA primers Cattle-1 (amplification length: 214bp) were designed to specifically identify cow feces. ), design the forward primer as shown in SEQ ID NO:1, and the reverse primer as shown in SEQ ID NO:2.

Embodiment 2

[0030] Embodiment 2 monitors pollution source

[0031] 1) DNA extraction:

[0032] DNA was extracted from 224 stool samples of 6 species (MO-BIO DNA Isolation kit);

[0033] 2) Specificity verification:

[0034] Get the genomic DNA in step 1), mix the DNA samples of the same species, respectively Cattle-1, FUP (universal primer, forward primer as shown in SEQ ID NO:3, reverse primer as shown in SEQ ID NO:4 Shown) as primers, add 7.5 microliters of TaqPCR Master Mix (2×), 0.5 microliters of forward primers, 0.5 microliters of reverse primers, and 1 microliters of genomic DNA; and add sterile water to 15 microliters; PCR reaction , the reaction temperature is as follows

[0035] Cattle-1: Pre-denaturation at 96°C / 300s, 1 cycle; denaturation at 96°C / 30s, annealing at 56°C / 30s, 32 cycles.

[0036] Such as figure 1 As shown in the primer specificity diagram, primer Cattle-1 reacts with Faecalibacterium in cow feces to produce bands, and there is no band in other species, in...

Embodiment 3

[0037] Example 3 Positive Rate Determination

[0038] DNA was extracted from 48 cow feces samples for detection, such as image 3 Among the 48 DNA samples shown, 44 samples can be PCR amplified by primer Cattle-1 and obvious bands can be seen, and the positive rate is 91.7%, which shows that the primer Cattle-1 has good stability.

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PUM

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Abstract

The invention belongs to technical detection of biotic environments, and particularly relates to a gene marker for monitoring a water pollution source in real time and application of the gene marker as well as a kit for monitoring cow dung pollution in water. The gene marker comprises a pair of primers, a positive primer of the primers is shown in SEQ ID NO:1, and a reverse primer of the primers is shown in SEQ ID NO:2. The gene marker can be specifically bound to a cow dung extract in the water, can rapidly determine pollution caused by cow dung in the water in real time, has the lowest detection limit of 3134 copies / [mu]L, has excellent sensitivity effects and has a positive rate up to 91.7% in same-species detection.

Description

technical field [0001] The invention belongs to bio-environmental technology detection, and in particular relates to a gene marker for real-time monitoring of water body pollution sources and its application, and a kit for monitoring cow dung pollution in water bodies. Background technique [0002] Cattle are one of the most widely distributed and stocked large livestock in the world, and beef is also one of the meat varieties with the largest audience. Due to the increasing demand for beef in the international market and the continued tight beef market, the number of live cattle in the world is on the rise. The number of live cattle in 2010 was 1.012 billion, and in 2013 it has increased to 1.027 billion. The increase in the number of live cattle has brought about a serious problem of cow manure pollution, and the drinking water pollution caused by its manure has brought great harm to human health and the natural environment. [0003] At present, the biological indicators ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 唐闻生王玥鉴柴宏祥张坤何强
Owner CHONGQING UNIV
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