Polymerase chain reaction (PCR) chip method for detecting transgenic components
A genetically modified component and chain reaction technology, applied in the field of polymerase chain reaction chip method, can solve the problems of low detection efficiency, achieve high sensitivity, high accuracy, and reduce mutual interference
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Embodiment 1
[0024] Embodiment 1PCR-chip sensitivity verification
[0025] As shown in Table 1, all primers were designed and synthesized for use. Soak the glass slide used as a chip in 5N NaOH for 2 hours; ddH 2 Rinse with O for 30 seconds; soak in 10% 3-aminopropyl-triethoxysilane (3-aminopropyl-triethoxysilane) aqueous solution at 80°C for 2 hours; rinse with ddH2O for 30 seconds; dry at 120°C for 1 Hours; 100 μl of 0.2 mg / ml neutravidin was dropped on the glass slide, placed in a humid chamber at room temperature for 2 hours; ddH 2 O rinse for 30 seconds; centrifuge to dry, dry and store for later use. Put downstream primers of appropriate concentration through the spotting instrument, and spot on the glass slide as required, which is the required chip.
[0026] Take the prepared samples 1, 2, 3 (Table 2) for PCR-chip reaction. Prepare the PCR reaction system according to Table 3, add 1 μl each of samples 1, 2, and 3 to the three PCR reaction systems, and perform PCR reactions in t...
Embodiment 2
[0033] Embodiment 2PCR-chip accuracy and high-throughput verification
[0034] The chip and primers were prepared as in Example 1. The sample is for corn-based feed. Get 100mg feed, extract high-quality DNA (DNA concentration 78.28ng / μl, OD 260 / OD 280= 1.68). Prepare the PCR reaction system according to Table 3. Since the known sample is corn, the selection of upstream primers is different from Example 1. The primer ZEIN of the endogenous gene of corn is selected. At the same time, soybean and rapeseed are selected to verify whether other raw materials are contained. , endogenous primers for potato and rice, Lectin, PE3-PEPase, PATA and GOS9; primers for exogenous genes CaMV 35s, ActinI, FMV 35s, NOS, PAT, Bar, GOX, CryIA(b), Cry9c, EPSPS; and Primers MON810, BT176, BT11, GA21, T25, TC1507, MON863, NK603, CBH351 related to the identification of transgenic maize lines. After adding the sample, carry out PCR reaction, the reaction program is: 95°C, 1min; 40 cycles (95°C, 3...
Embodiment 3
[0035] Example 3 PCR-chip high-throughput verification
[0036] The chip and primers were prepared as in Example 1. The sample was a mixture containing rice, rapeseed, and potato. A 100 mg sample was taken and DNA was extracted (DNA concentration 58.04 ng / μl, OD260 / OD280=2.07). Prepare the PCR reaction system according to Table 3, select species identification gene primers Lectin, ZEIN, GOS9, PE3-PEPcase, PATA; exogenous genes CaMV35s, Actin I, FMV 35s, NOS, PAT, Barnase, Barstar, Bar, NPTII, GOX, Primers for EPSPS, Cry1Ab / c, Cry3A, PVY-cp, PLRVrep; and primers related to the identification of related transgenic plant lines Bt63, Topas19 / 2, RF3, OXY235, Ms8, NewLeaf Atlantic, NewLeafY Russet Burbank, NewLeaf Y Shepody, NewLeaf Plus Russet Burbank, EH92-527. After adding the sample, carry out PCR reaction, the reaction program is: 95°C, 1min; 40 cycles (95°C, 30S; 56°C, 150S; 72°C, 60S); 72°C, 10min. After the PCR reaction, the chip was rinsed, dried and scanned for detecti...
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