Coding gene SOD in blood related to dairy cow ketosis and PCR detection kit thereof
A detection kit and coding gene technology, which can be used in genetic engineering, plant genetic improvement, microbial measurement/inspection, etc., can solve the problems of cumbersome methods, unstable results, time-consuming, etc., and achieve good repeatability
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Embodiment 1
[0022] Example 1 illustrates the bioinformatics method to identify the distribution of SOD genes.
[0023] At NCBI, use the Blastn online comparison software (http: / / blast.ncbinlm.nih.gov / Blast.cgi) to search for the SOD gene in the genome-wide database, and the search results show that the cow body contains the SOD gene (SEQ ID NO.1 ), and the nucleotide sequence with a gene length of 134bp is reverse-transcribed into cDNA after extracting RNA to detect the concentration and quantify it. The expression level of SOD gene in the blood of healthy dairy cows is extremely high; the expression level of SOD gene in the blood of ketosis cows is extremely low ; Both bands can be seen at 143bp. The results of bioinformatics identification showed that SOD had good specificity and could be used to establish a PCR method for rapid detection of SOD in dairy cow blood.
Embodiment 2
[0025] This example illustrates the preparation of the kit.
[0026] Primer design and synthesis: Using the SOD gene as a template, design and analyze primers, and select the best detection primers according to the genomic DNA sequence. The nucleotide sequences are shown in Table 1 below:
[0027] Table 1 Primer Sequence
[0028]
[0029] The above-mentioned primer pairs can be packaged individually, or can be made into a PCR detection solution. In the PCR detection solution, the amount of the above-mentioned primer pairs can be the conventional amount known to those skilled in the art.
[0030] That is to say, the kit of the present invention may contain the aforementioned independently packaged primer pair, or may contain a configured PCR detection solution containing the primer pair.
[0031] Further, the kit may also contain double distilled water (ddH20), 2XTaq Master Mix and the like.
Embodiment 3
[0033] This example illustrates the use of the kit.
[0034] (1) Extract genomic RNA from the sample; detect the concentration of RNA in the extracted sample, quantify it and reverse transcribe it into cDNA. The steps of extracting sample RNA are as follows: (a) take the processed tissue, feces supernatant and blood, and add Trizol lysate for lysing. The liquid in the 2mL EP tube was mixed evenly, shaken vigorously with a vortex shaker, and placed on ice for 10 min. (b) Add 200 uL of chloroform, shake the EP tube vigorously for 1 min, so that it can extract nucleic acid better, and let it stand on ice for 10 min. (c) Centrifuge at 12000r / min for 15min, extract 2 / 3 of the supernatant of the transparent liquid in the test tube, add an equal amount of isopropanol and mix gently. Let stand on ice for 10 minutes. (d) The centrifugal time of 12000r / min is 10min, and the configuration of 75% absolute ethanol (mixed with DEPC water) is carried out. (e) Discard the supernatant, add...
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