Establishment of SHERLOCK-HBA Detection Method and Its Application in Blood Identification
Objective Rapid and accurate identification of body fluid traces at crime scenes is crucial for case investigation.Leveraging the speed and sensitivity of nucleic acid detection technology based on SHERLOCK,our research focuses on developing a peripheral blood SHERLOCK-HBA detection system to detect mRNA in forensic practice.Methods Short crRNA fragments targeting the blood-specific mRNA gene HBA were designed and screened,alongside RPA primers.Optimal RPA primers were selected based on specificity and amplification efficiency,leading to the establishment of the RPA system.The most efficient crRNA was chosen based on relative fluorescence units(RFU)generated by the Cas protein reaction,and the Cas protein reaction system was constructed to establish the SHERLOCK-HBA detection method.The RPA and Cas protein reaction systems in the SHERLOCK detection system were then individually optimized.A total of 79 samples of five body fluids were tested to evaluate the method's ability to identify blood,with further verification through species-specific tests,sensitivity tests,mixed spots detection,aged samples,UV-irradiated samples,and actual casework samples.Results The SHERLOCK reaction system for the peripheral blood-specific marker HBA was successfully established and optimized,enabling detection within 30 min.The method demonstrated a detection limit of 0.001 ng total RNA,better than FOB strip method and comparable to RT-PCR capillary electrophoresis.The system could detect target body fluids in mixed samples and identify blood in samples stored at room temperature for three years and exposed to UV radiation for 32 h.Detection of 11 casework samples showed performance comparable to RT-PCR capillary electrophoresis.Conclusion This study presents a CRISPR/Cas-based SHERLOCK-HBA detection system capable of accurately,sensitively,and rapidly identifying blood samples.Introducing CRISPR/Cas technology to forensic body fluid identification represents a significant advancement in applying cutting-edge molecular biology techniques to forensic science.The method's simplicity,shorter detection time,and independence from specialized equipment make it promising for rapid blood sample identification in forensic cases.
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维普
