There are a few different ways that you can check the specificity of the primers that you have designed for infusion cloning:
BLAST: One option is to use the Basic Local Alignment Search Tool (BLAST) to search for sequence homology between your primers and the genome of the organism you are targeting. This can help you to determine whether your primers are specific to the desired region of the genome, or if they may bind to other non-targeted regions as well.
Multiple alignment: You can also perform a multiple sequence alignment of your primers with the target genomic region to visualize the binding sites of your primers. This can help you to identify any potential off-target binding sites that your primers may have. Personally, I use Mega Software.
Primer-BLAST: The Primer-BLAST tool is a specific tool that allows you to design and validate primers for PCR and other molecular biology applications. It can help you to identify potential off-target binding sites and evaluate the specificity of your primers.
In silico PCR: In silico PCR is a computational tool that allows you to simulate PCR reactions using a genomic sequence as a template. You can use in silico PCR to test the specificity of your primers and determine whether they will amplify the desired target region.