NASBA-ELISA rapid detection kit and detection method for anti-glufosinate resistance gene component in transgenic corn
A technology for detection kits and detection methods, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high cost of gene chip technology, complicated data analysis, loss of antigenicity, etc., and achieve easy promotion and repeatability. Good sex and good effect
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example 1
[0072] Example 1: NASBA-ELISA kit for specific detection of glufosinate-resistant bar gene in maize:
[0073] Extract the RNA of transgenic bar gene corn, transgenic Badh drought-resistant gene corn, Cry insect-resistant gene corn, and non-transgenic corn, respectively, take 0.5 μL RNA respectively and 10 μL NASBA amplification reaction solution, heat at 65 °C for 2 min, and then cool at 41 °C for 2 min; Immediately add 5 μL of enzyme mixture and flick to mix well, ddH 2 O to make up the reaction volume of 20 μL, react at 41°C for 90 min, stop the reaction in ice bath, and perform ELISA detection with NASBA product detection reagent.
[0074] The results showed that only the total RNA amplification product of transgenic maize leaves had the target band with the expected size of 519bp, the rest of the transgenic maize and non-transgenic maize had no amplification signal, and the negative control had no signal, indicating that the kit has no effect on bar RNA Assays are specifi...
example 2
[0075] Example 2: sensitivity test of bar-NASBA kit:
[0076] Extract the total RNA of the transgenic maize, and serially dilute the RNA with an initial concentration of 500ng / μL bar. For each gradient, take 0.5μL template RNA and 10μL NASBA amplification solution, heat at 65°C for 2min, cool at 41°C for 2min; add 5μL enzyme The mixture was mixed well, ddH 2 O to make up 20μL volume, react at 41°C for 90min, stop the reaction in ice bath, detect NASBA products by ELISA, the results show that the RNA was diluted to 10 -8 There are still positive amplification products, the sensitivity is 5fg, and the negative control has no detection signal, indicating that the sensitivity of the kit can reach 5fg, which has high sensitivity, as shown in Table 1.
[0077] Table 1 Bar-NASBA kit sensitivity test
[0078]
[0079]
[0080] Electrophoresis results also showed that the detection concentration can reach 1×10 -8 concentration, the concentration is 1×10 -9 When there is no de...
example 3
[0081] Example 3: Sample detection of maize glufosinate resistance gene bar-NASBA kit
[0082] (1) Preparation of sample RNA
[0083] (a) Take 120mg each of non-transgenic and transgenic corn leaves, corn stems and corn roots, grind with liquid nitrogen, add 1mL Trizol, vortex for 1min, and place at room temperature for 15min.
[0084] (b) Add 500 μL of chloroform, invert and mix several times, leave at room temperature for 5 minutes, and centrifuge at 12000 r / min for 15 minutes at 4°C.
[0085] (c) Add an equal volume of isopropanol to the supernatant, bathe in ice for 20 min, centrifuge at 10 000 r / min for 10 min at 4°C, and remove the supernatant.
[0086] (d) The precipitate was washed twice with an appropriate amount of 75% ethanol, and centrifuged at 10 000 r / min for 5 min at 4°C.
[0087] (e) After natural drying, add 50 μL of DEPC-treated TE to redissolve, and identify by ultraviolet spectrophotometry and electrophoresis.
[0088] (2) NASBA amplification reaction pr...
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