ELISA (enzyme-linked immunosorbent assay)
ELISA possesses the high sensitivity of enzyme reaction and the specificity of antigen-antibody reaction, and has the characteristics of simplicity, rapidity, and low cost, etc. However, it is prone to the problem of too high background and can only detect the crude products whose antigenicity of target proteins has not been significantly changed. The direct method and double antibody sandwich method both need to prepare specific enzyme-labeled antibody, the preparation method is more cumbersome, and an enzyme-labeled antibody can only detect one kind of protein, while the enzyme-labeled anti-body applicable to the indirect method has been sold commercially. Whether the detection kit for a transgenic protein can be generalized among different plants expressing the same exogenous protein has to be tested.
Lateral Flow Immunoassay:
The "lateral flow" type of immunoassay has been developed in the last 15 years, and was previously used primarily in the medical field. Similar to ELISA, this assay is based on the principles of the sandwich technique, but the assay is performed on a solid-phase support rather than in a tube, and the labeled antigen-antibody complex migrates sideways until it encounters an antibody on a stationary surface. Kits for lateral flow immunoassays are now commercially available and can be used for field testing. Sample handling for this method is simple compared to DNA-based assays. Since the target protein is generally water-soluble and the antibody is highly specific, the test sample only requires preliminary processing to meet the test requirements, which is fast, easy and suitable for field operation. Lateral flow immunoassay test strip is a very fast and simple qualitative detection method, the test strip is placed in the sample extract to be tested, the results can be obtained in 5 to 10 minutes, without the need for special equipment and skilled skills. However, a test strip can only detect one protein, and can only detect the presence or absence of exogenous proteins but cannot distinguish between specific transgenic varieties. Lateral flow immunoassays are either qualitative or semi-quantitative. Following the appropriate sampling procedure, less than 0.15% of the GM content can be detected in a given batch of samples at a 99% confidence level.
Advantages and Disadvantages of Common Polymerase Chain Reaction (PCR)
Advantages:
1) Higher accuracy
2) Higher sensitivity
3) Lower price
Disadvantages:
1) Complexity of the procedure requires a certain background
2) Dyes used in the electrophoresis can be harmful
3) Dye used in the electrophoresis can be harmful to humans. Hazardous
3)Only one indicator can be detected
Advantages and disadvantages of real-time fluorescence quantitative PCR (qPCR)
Advantages
1)High sensitivity
2)High accuracy
3)High throughput
4)Real-time monitoring can be realized
5)Quantitative judgement can be realized
Disadvantages
1)High price
2)Complicated operation
3)Few supporting products
4)High maintenance cost
Constant Temperature Fluorescence PCR Detection Methods:
Constant Temperature PCR technology can be summarized as being more accurate, rapid, and easy to implement than PCR. Because of the realization of constant temperature amplification, special primer design, making the detection more accurate; no denaturation, annealing and other links, the whole process is in the replication of DNA fragments, there is no time loss, so that the detection is more rapid; no thermal cycling needs, simple equipment, easier to promote the application. It creates a brand new space for the promotion and application of PCR technology. At present, the mainstream real-time constant temperature fluorescence PCR instruments are Guangzhou DIAO biological Deou-308C constant temperature fluorescence detector, 3M Molecular Detection System, Optigene Genie II and Genie III, Qiagen TuberScaner II, Envirologix DNAble, etc.