Nested PCR (polymerase chain reaction) kit for detecting duck-manure pollution in water body and detection method thereof
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A kit and duck manure technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of untraceable methods for detecting fecal contamination, and achieve good specificity and high sensitivity , the effect of high application value
Active Publication Date: 2015-09-02
NANJING UNIV
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[0007] Aiming at the technical problem that the traditional method for detecting fecal pollution cannot be traced back, the present invention provides a nested PCR detection method and kit for detecting duck feces pollution in water bodies, which can quickly and accurately detect trace amounts of duck feces pollution in water bodies
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Embodiment 1
[0036] Nested PCR kit for detecting duck manure pollution in water, including two pairs of nested PCR primers, dNTP, PCR buffer, Taq enzyme, Mg 2+ and ddH 2 O, two pairs of nested PCR primers are:
[0037] SDF: 5’-GCATAGGGCAACACGGAAAG-3’
[0038] SDR: 5’-GTTCTGGGTGTTGTCCGGTA-3’
[0039] NDF: 5’‐CCACGTAAATGCCAAAGATG‐3’
[0040] NDR: 5'‐CTGAGCTAGAGGAAGGCT‐3'. The above two pairs of nested PCR primers were custom-synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0041] ddH above 2 O is sterile double distilled water. The concentration of Taq enzyme is 5U / ul. Mg 2+ The concentration of the solution is 25mmol / L, which is MgCl 2 solution. The concentration of dNTPs was 2.5 mmol / L. PCR buffer is 10×PCR buffer (purchased from Takara, Code No.RR001A)
[0042] Collect fresh chicken, human, pig, cow, sheep, duck, goose, and mouse feces, extract DNA from 0.2 grams of feces, and use it as a template for the first round of PCR amplification. The reaction system is 2.5ul ...
Embodiment 2
[0047] Take 0.2 grams of duck feces to extract DNA, and the measured DNA concentration is 79.2ng / ul. The DNA solution is serially diluted by four-fold dilution method, and the dilution times are 1 / 4 and 1 / 4 respectively. 2 , 1 / 4 3 , 1 / 4 4 , 1 / 45, 1 / 46, 1 / 47, 1 / 48, and 1 / 49 duck feces DNA solutions, respectively take the DNA solutions of various concentrations as templates for the first round of PCR amplification, and the reaction system is 2.5ul of 10 ×PCR buffer; 2.5ul dNTP; 0.1ul Taq enzyme; 2ul of 25mmol / L Mg 2+ Solution; 0.5ul concentration of 10umol / L upstream and downstream primers SCF, SCR; 2ul template DNA; sterilized ddH 2 Make up to 25ul with O; the reaction conditions are 95°C, pre-denaturation for 5min; 40 cycles of 95°C for 30s, 57°C for 30s, 72°C for 45s; 72°C for 7min, and storage at 4°C.
[0048] Perform 1% agarose gel electrophoresis on the first round of PCR products, the electrophoresis conditions are: voltage 120V, current 440mA, time 25min, the electrop...
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Abstract
The invention discloses a nested PCR (polymerase chain reaction) kit for detecting duck-manure pollution in a water body and a detection method thereof, and belongs to the field of the detection of pollution in water bodies. The nested PCR kit for detecting the duck-manure pollution in the water body comprises two pairs of nested PCR primers, dNTP (deoxyribonucleoside triphosphate), a PCR buffer, a Taq polymerase, a Mg<2+> solution and ddH2O (double distilled water). Two rounds of PCR amplification are used by a nested PCR method. The detection method comprises the following steps of extracting a DNA (deoxyribonucleic acid) in a to-be-detected water sample, and carrying out a first round of PCR amplification by using the DNA in the to-be-detected water sample as a template, wherein the primers are an SDF (stroma derived factor) and SDR (short-chain dehydrogenase / reductase); after a first reaction is terminated, carrying out a second round of PCR amplification by using a product of the first round of PCR amplification as a template, wherein the primers are an NDF (neutral detergent fiber)and NDR, and after a second reaction is terminated, detecting the existence of a 158bp strip, which shows that the water body is subjected to the duck-manure pollution, through agarose gel electrophoresis. According to the method, the detection sensitivity is greatly increased through nested PCR amplification; a little duck-manure pollution which exists in water can be detected; moreover, the method has a favorable specificity, and can be directly applied to the detection and the preventive treatment work of fecal pollution in the water body.
Description
technical field [0001] The invention relates to the field of water body pollution detection, in particular to a nested PCR kit for detecting duck manure pollution in feces-polluted water bodies and a detection method thereof. Background technique [0002] In recent years, my country's water pollution has become increasingly serious, threatening people's living environment and healthy water use, and causing serious economic losses to the society. The livestock and poultry breeding industry in the Taihu Lake Basin is well developed, and the breeding wastewater is one of the main sources of water pollution in the Taihu Lake Basin. When fecal pollutants enter the water body, it will not only increase the nitrogen and phosphorus content of the water body, causing eutrophication, but also bring in the pathogenic bacteria that may exist in the feces, causing the water transmission of diseases (Baldursson, S. and Karanis, P. (2011) Waterborne transmission of protozoan parasites: Re...
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