Rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and real-time PCR.

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Crime scene investigations face constraints in analyzing biological evidence.
• Conventional ABO blood group identification is limited to blood and not suitable for other bodily fluids.
• Existing ABO genotyping methods are time-consuming, costly, and require advanced equipment.

Purpose of the Study:

• To develop rapid and efficient ABO genotyping methods for forensic applications.
• To overcome limitations of conventional serological and SNP-based ABO identification.
• To enable faster screening of biological evidence at crime scenes.

Main Methods:

• Developed a rapid ABO genotyping method utilizing loop-mediated isothermal amplification (LAMP).
• Employed multiplex real-time polymerase chain reaction (PCR) for ABO genotyping.
• Selected three single nucleotide polymorphism (SNP) sites (nt 261, 526, 803) in the ABO gene for analysis.
• Validated genotyping results using DNA sequencing of saliva samples and mock forensic samples (cotton swab, filter paper).

Main Results:

• Successfully identified six ABO genotypes within 30 minutes using LAMP at a constant 65°C.
• Multiplex real-time PCR enabled detection of major ABO groups and subgroups (e.g., cis-AB).
• Amplification results from both LAMP and real-time PCR methods showed high concordance with sequencing data.

Conclusions:

• The developed LAMP and multiplex real-time PCR methods provide rapid and accurate ABO genotyping.
• These methods offer an efficient way to screen biological evidence at crime scenes.
• The techniques can significantly aid in identifying investigative leads in criminal cases.