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Principle and method application of rapid detection of food microorganisms based on PCR technology
The safety of agricultural products and food caused by food-borne microorganisms has attracted more and more attention
The polymerase chain reaction (PCR) method is applied to microbial testing, and has outstanding advantages such as rapidness, strong specificity, and high sensitivity, so it has attracted more and more attention from the academic community
(1) Principle of PCR amplification
PCR (Polymerase Chain Reaction), or polymerase chain reaction, is an in vitro rapid DNA amplification method pioneered by Kary Mullis et al.
(2) PCR reaction system
The PCR reaction is to put a mixture of template DNA, primers, Taq enzyme, dNTPs, magnesium ions, buffer, double distilled water, etc.
Template DNA: PCR template can be single-stranded or double-stranded DNA.
Primer: A single-stranded oligonucleotide fragment complementary to both sides of the DNA fragment to be amplified, usually 15-25 bases in length
①The GC content of the primers is preferably 45%~55%, and the GC should be randomly distributed to avoid the accumulation of purines and pyrimidines ;
②The hairpin structure should not be formed inside the primer;
③Two kinds of primer T, the value should be as close as possible;
④The two primers cannot be complementary, especially near the 3'end
DNA polymerase: High temperature DNA polymerase Taq is often used in PCR reactions
dNTPs: are the "raw materials" for PCR amplification, consisting of 4 deoxynucleosides (dATP, dGTP, dTTP, dCTP) at the same concentration
Mg²+: a metal ion necessary for Taq enzyme activity
Related links: rapid detection of microbes in bulk ready-to-eat food