The coupling of methylation-sensitive restriction enzyme with recombinant polymerase amplification and lateral flow dipstick for the detection of (menstrual) blood stains

Blood is a critical and frequently encountered type of evidence at forensic crime scenes. Its detection can provide valuable insights into the nature of a case and help refine investigative focus. The blood test strip is the most commonly used method for blood detection. However, it is prone to false-negative results, especially when detecting trace amounts of blood found at crime scenes, as well as challenging samples, such as those buried in soil, washed with detergents, and aged samples. In this study, we developed an alternative (menstrual) blood detection method based on the blood-specific methylation site (cg04011671). After treatment with a Methylation-Sensitive Restriction Enzyme (MSRE), the DNA is then amplified using Recombinant Polymerase Amplification (RPA), and the amplification is visualized through Lateral Flow Dipstick (LFD). Our results demonstrate that the MSRE-RPA-LFC method exhibits good specificity for body fluids and species. Additionally, this method shows a sensitivity of 125 pg DNA input, indicating its high detection capability for trace samples. Furthermore, this method is compatible with subsequent DNA analysis and demonstrates superior detection performance compared to traditional blood test strips (e.g., FOB test strips). It can effectively detect samples buried in soil for two weeks or longer, samples cleaned with reagents (e.g. hydrogen peroxide or oxygen bleach), and aged samples stored at room temperature for 10, 9, and 6 years. These findings indicate that this method serves as a confirmatory approach for (menstrual) blood identification and is a promising technique for detecting (menstrual) blood in trace and challenging forensic samples.