Abstract: Disclosed is a hot-start PCR method, based on protein trans-splicing of intein-inserted large (Neq L) and small (Neq S) fragments of Neq DNA polymerase. The method comprises: preparing a PCR reaction mixture containing a sample DNA and primers; adding the Neq L fragment and the Neq S fragment together to the PCR reaction mixture, said Neq L fragment consisting of an amino acid sequence of SEQ ID NO: 2, with an intein amino acid sequence stretching from position 579 to 676 therein, said Neq S fragment consisting of an amino acid sequence of SEQ ID NO: 4 with an intein amino acid sequence stretching from position 1 to 30 therein; inducing the Neq L fragment and the Neq S fragment to undergo a protein trans-splicing process to form a polypeptide exhibiting Neq DNA polymerase activity; and performing a certain number of cycles of DNA denaturation, primer annealing and DNA extension.