Here at TwistDx™ we are constantly working to improve RPA even further, and are discovering new capabilities and features of the reaction. We can now report that in some instances RPA will work straight out of the box with established oligonucleotide PCR primers that are as short as 18 residues, and which require no additional optimisation. To date, we have found that PCR primers will work for amplicons of up to 500 base pairs in length, although incubation times do vary with primer length and amplicon size for gel-based detection.
Primer sequences
C. t1 TCTTTTTAAACCTCCGGAACCCACTT/GGATGGCATCGCATAGCATTCTTTG
C. t2 GGACAAATCGTATCTCGG/GAAACCAACTCTACGCTG
C. t3 TGATGCTAGGGACGGATTAAAACC/TTCCCCTAAATTATGCGGTGGAA
H. s1 CGTGTACTTGATCGTCGCCAT/CAAGGAGGAAGGCTAGGGCTA
H. s2 GTGCTGTTTGCAGCTCTGAG/AGTGGCCAAGGAGTCGTATG
N. g1 CCGGAACTGGTTTCATCTGATT/GTTTCAGCGGCAGCATTCA
N. g2 GCTACGCATACCCGCGTTGC/CGAAGACCTTCGAGCAGACA
T. v ATTGTCGAACATTGGTCTTACCCTC/TCTGTGCCGTCTTCAAGTATGC
References:
C. t1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854900/
C. t2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC269699/
C. t3 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944303/
H. s1 http://pga.mgh.harvard.edu/primerbank/[SK2]
H. s2 http://www.ncbi.nlm.nih.gov/pubmed/12209956
N. g1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876173/
N. g2 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC495310/
T. v http://www.ncbi.nlm.nih.gov/pmc/articles/PMC87441/