Genetically modified rice PA110-15 transformant specificity quantitative PCR detection primer
A technology for transgenic rice and detection primers, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve problems such as the inability to achieve quantitative detection.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0032] Example 1 Extraction and Detection of Plant Genomic DNA
[0033] Plant genomic DNA extraction kit DP305 (Tiangen Biochemical Technology Co., Ltd.) was used to extract and purify PA110-15 rice genomic DNA and common rice genomic DNA. The extraction method is as follows:
[0034] 1) Take 200mg rice powder sample;
[0035] 2) Add 800 μL of 65°C preheated GP1 buffer, quickly invert and mix well, place the centrifuge tube in a 65°C water bath for 1 hour, and invert the centrifuge tube during the water bath to mix the sample several times.
[0036] 3) Add an equal volume of phenol:chloroform (1:1), mix well, and centrifuge at 12000rpm for 10min.
[0037] 4) Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix thoroughly, and centrifuge at 12000 rpm for 10 min.
[0038] 5) Take the supernatant, add an equal volume of GP2, and mix well.
[0039] 6) Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000 rpm for 30...
Embodiment 2
[0046] Example 2 standard curve drawing
[0047] The standard curves of PA110-15 transformants and PLD internal standard genes were drawn using the same standard samples. Dilute the extracted PA110-15 rice genomic DNA with 0.1×TE gradient to 50ng / μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL, 0.005ng / μL, in the real-time fluorescent PCR reaction system, take 2μL As a template, the corresponding copy numbers 212766, 21276, 2127, 212, 21 were prepared to draw the standard curve.
[0048] Transformant-specific quantitative primers and probes were designed according to the sequence of the junction region of the inserted gene carrier of PA110-15 and its 3' side sequence as the target sequence, and PLD was used as the internal standard reference gene, and the PLD amplification method was quoted from the EU standardization Detection method "Event-specific Method for the Quantitation of RiceLineLLRICE62Using Real-timePCR–ValidationReportandProtocol-SamplingandDNAExtractionofRice". Quantitative ...
Embodiment 3
[0070] The accuracy experiment that embodiment 3 detects
[0071] Divided into 4 experimental groups, experimental group 1 mixed 40g transgenic PA110-15 rice seeds and 10g non-transgenic common rice seeds; experimental group 2 mixed 25g transgenic PA110-15 rice seeds and 25g non-transgenic common rice seeds; experimental group 3 Mix 20 g of transgenic PA110-15 rice seeds with 30 g of non-transgenic common rice seeds; Experimental group 4 mixes 10 g of transgenic PA110-15 rice seeds with 40 g of non-transgenic common rice seeds. According to the method of Example 1, the genomic DNA of the above-mentioned experimental group was extracted, and the sample was diluted to 0.7ng / μL. According to the PCR system described in Example 2, fluorescent quantitative PCR was performed, and the Ct amplified by the specific real-time fluorescent PCR reaction system was read. (Ct value and the Ct value of standard gene real-time fluorescence PCR reaction system amplification in rice, calculate...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More