A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

• We developed PNA probe-based FMCA for ABO genotyping.
• This method allowed for simultaneous detection of three SNP sites in the ABO gene.
• Eighty samples were successfully genotyped.
• Reactions were performed in one single closed tube in less than 3 h.
• This method may be an effective and helpful tool in forensic investigations.

ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n = 5], A/O01 or A/O02 [n = 23], B/B [n = 3], B/O01 or B/O02 [n = 18], A/B [n = 9], O01/O01 or O01/O02 or O02/O02 [n = 20], cis-AB01/A [n = 1], and cis-AB01/O01 or cis-AB01/O02 [n = 1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3 h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.