Pig manure contaminated specific molecular marker 3-53 based on host-enteric microorganism interacting genes and detecting method for pig manure contaminated specific molecular marker 3-53
A technology of intestinal microorganisms and molecular markers, which is applied in the field of pig fecal pollutant detection, can solve the problems of high misjudgment rate, insufficient host specificity, and insufficient accuracy of animal sources, etc., and achieve high sensitivity and strong specificity
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Embodiment 1
[0023] Example 1 Enrichment screening of specific pig-gut microbe interaction genes
[0024] 1.1 A total of 145 feces samples from 6 species were collected, including 34 swine feces samples, 20 cow feces samples, 12 goat feces samples, 8 sheep feces samples, 7 chicken feces samples, 20 duck feces samples, Five goose feces samples and 13 human feces samples were collected. Place the sample in a sterile container, add 3mL GITC buffer (5M guanidine isothiocyanate, 100Mm EDTA (pH 8.0), sodium lauryl sarcosine) to every 200mg (wet weight) sample, and store it at low temperature. DNA extraction from feces Fast DNA Stool Kit (Qiagen, Valencia, CA) instructions. And its concentration and purity were determined on a UV spectrophotometer NanoDrop ND-2000UV (NanoDrop Technologies, Thermofisher).
[0025] 1.2 Host (pig) specific genome enrichment (GFE)
[0026] Genomic DNA of all single pig source samples was mixed to form a genomic mixed library to increase the diversity of the gen...
Embodiment 2
[0043] Example 2 Screening and reaction conditions of molecular markers
[0044] A porcine-specific DNA gene library was established by competitive hybrid gene fragment enrichment (GFE). After the library was cloned into pCR TOPO 4.0, 500 clones were randomly selected for sequencing analysis, and a total of 382 non-redundant porcine-specific sequences were obtained. BLAXTS analysis found that 60 non-redundant sequences were related to host-microbe interaction proteins such as surface proteins such as Bacteroidetes and Clostridials, membrane secreted proteins, and carbohydrate metabolism proteins. As a target for porcine-specific molecular marker screening. Molecular markers were designed for these 60 genes, and after screening, two sets of molecular markers were found to be missing from pig-derived fecal contamination (Table 1).
[0045] Table 1 Molecular marker sequence
[0046]
[0047] Using positive DNA as a template, repeated experiments found that when the concentr...
Embodiment 3
[0050] Example 3 standard curve and system repeatability
[0051] Dilute the two positive DNA standards 1-38 and molecular marker 3-53 10 times to 1.2×10 8 to 1.2×10 1 After the copy / μl, the fluorescent PCR reaction was carried out separately, and each dilution was repeated three times, and the standard curve was drawn to calculate the amplification efficiency. The results showed that the two sets of molecular markers had wide amplification linear range and high amplification efficiency, which were 1.07 and 0.95 ( image 3 and Figure 4 ). At the same time, the minimum detection limits of molecular markers 1-38 and 3-53 were 600 copies / reaction and 60 copies / reaction, respectively, indicating that both detection systems had good detection sensitivity.
[0052] Perform 10 repeated detections on the two positive standard DNAs with multiple dilutions by fluorescent PCR. The standard deviations of the Ct values of the molecular marker 1-38 and 3-53 reaction systems were betw...
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