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A primer, kit and high-throughput traceability method for detection of fecal pollution in water bodies

A pollution detection, high-throughput technology, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Sensitivity, time and cost savings

Active Publication Date: 2020-06-09
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the problems of many types of potential pollution sources and difficult identification of feces pollution in water bodies, the present invention designs a nested PCR general primer capable of amplifying multi-species species-specific mitochondrial DNA fragments, and provides a combination of nested PCR and Qualcomm High-throughput fecal (human, livestock, wild birds, mammals, etc.) pollution traceability method based on quantitative sequencing technology can achieve the purpose of rapid and accurate investigation and detection of fecal pollution sources in water bodies

Method used

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  • A primer, kit and high-throughput traceability method for detection of fecal pollution in water bodies
  • A primer, kit and high-throughput traceability method for detection of fecal pollution in water bodies
  • A primer, kit and high-throughput traceability method for detection of fecal pollution in water bodies

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Embodiment 1

[0042] The present invention can simultaneously detect fecal pollution including sources such as human beings, livestock and poultry, wild birds and mammals, and the steps are as follows: figure 1 shown. In this embodiment, people, pigs, cattle, sheep, chickens, ducks, geese, cats, mice, and Grackles were selected as representatives to simulate fecal contamination of various species in the laboratory to verify the applicability of the general primers of the present invention. .

[0043] Collect the fresh feces of the above species, take a small amount and add them to the river water not polluted by feces, take 1L each of the river water containing feces, filter them with a 0.22μm pore size mixed cellulose ester membrane, and use the FastDNA SPIN Kit for Soil (Takara) kit The DNA on the filter membrane was extracted and used as template DNA for nested PCR amplification. The PCR instrument was an ABI Veriti96 instrument. figure 2 The universal primer pair designed for the pre...

Embodiment 2

[0050] The detection of multiple fecal-polluted water bodies was simulated using feces of the above 10 species. Add 10 kinds of feces to the river water not polluted by feces at the same time, among which the amount of cow dung is the largest. Take 1L of river water, filter it with a 0.22μm pore size mixed cellulose ester membrane, and use the FastDNA SPIN Kit for Soil (MP bio) kit Extract the DNA on the filter membrane, and use this as template DNA to carry out nested PCR amplification, the method is the same as in Example 1. The nested PCR product was subjected to gel electrophoresis, and the method was the same as in Example 1. The PCR product at 500 bp was cut out, purified using the Takara MiniBEST Agarose Gel DNA Extraction Kit (Takara), and then high-throughput sequencing was performed using the Illumina Miseq sequencing platform. The long mode is 300bp at each paired end. The obtained sequencing data in .fastq format is firstly quality-controlled with prinseq-lite soft...

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Abstract

The invention discloses a primer, a kit and a high-throughput traceability method for detecting water body feces pollution. The detection range covers human, livestock and poultry and various wild animal feces pollution, and belongs to the field of water body pollution detection. It mainly includes the following steps: extracting the DNA of the water sample to be tested; using two pairs of universal primers for amplifying mitochondrial DNA to perform nested PCR amplification on the DNA of the water sample; performing high-throughput sequencing on the amplified product; combining the sequencing data with the mitochondrial DNA The database is compared and annotated, and the source of fecal pollution in the water samples is determined based on the comparison results. The present invention utilizes nested PCR technology to amplify mitochondrial DNA indicating fecal pollution in water samples, which has extremely high sensitivity. Using the universal primers of the present invention combined with high-throughput sequencing, not only can a variety of potential fecal contamination be simultaneously Qualitative traceability can also carry out relative quantification of the degree of various types of fecal pollution, so as to determine the main source of pollution.

Description

technical field [0001] The present invention relates to a method for quickly tracing the source of feces pollution in water bodies, more specifically, a method that uses mitochondrial DNA carried in animal feces as a marker, and uses nested PCR technology combined with high-throughput sequencing technology to simultaneously detect a variety of Methods for screening for potential fecal contamination. Background technique [0002] In recent years, with the continuous growth of population and economy, water pollution has become increasingly serious, and fecal pollution is particularly prominent. Fecal pollutants entering the water body will not only increase the nitrogen and phosphorus content of the water body, resulting in eutrophication of the water body, but also bring in the pathogenic bacteria that may exist in the feces, causing the spread of diseases in the water body. Therefore, it is urgent to prevent and control water body fecal pollution. [0003] There are many p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6848C12Q1/6869C12N15/11
CPCC12Q1/6888C12Q1/6848C12Q1/6869C12Q2600/16C12Q2549/119C12Q2531/113C12Q2535/122C12Q1/6806
Inventor 何席伟张徐祥高洁齐昭栋
Owner NANJING UNIV
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