SHERLOCK-HBA检测方法的建立及其在血液鉴别中的应用  

Establishment of SHERLOCK-HBA Detection Method and Its Application in Blood Identification

作　　者：姚乾威 何红霞 胡胜 赵一霞 罗雨 季安全 孙启凡 YAO Qian-Wei;HE Hong-Xia;HU Sheng;ZHAO Yi-Xia;LUO Yu;JI An-Quan;SUN Qi-Fan(Faculty of Forensic Sciences,Shanxi Medical University,Taiyuan 030001,China;MPS’s Key Laboratory of Forensic Genetics,National Engineering Laboratory for Crime Scene Evidence Investigation and Examination,Institute of Forensic Science,Ministry of Public Security(MPS),Beijing 100038,China;Beijing Institute of Microbiology and Epidemiology,Academy of Military Medicine,PLA Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]山西医科大学法医学院,太原030001 [2]公安部鉴定中心,法医遗传学公安部重点实验室,北京100038 [3]中国人民解放军军事科学院军事医学研究院微生物流行病研究所,北京100071

出　　处：《生物化学与生物物理进展》2024年第8期1971-1982,共12页Progress In Biochemistry and Biophysics

基　　金：国家重点研发计划(2022YFC3341002);公安部科技强警基础工作计划(2022JC11)资助项目。

摘　　要：目的基于规律成簇的间隔短回文重复(CRISPR)/CRISPR关联蛋白质系统(Cas)技术建立检测血液特异性血红蛋白α(HBA)mRNA的方法,以准确、快速地鉴定血液样本。方法本研究通过设计和筛选血液特异性HBA mRNA上的CRISPR RNA(crRNA)短片段,基于CRISPR/Cas技术原理建立了SHERLOCK-HBA检测方法,对5种体液(包括外周血、月经血、唾液、阴道分泌液、精液)共79份样本进行检测以评价所建方法鉴别血液的能力,并通过种属特异性实验、灵敏度实验、模拟混合斑检测、模拟降解样本检测、实际案件样本检测等进一步验证实验体系的法医学应用能力。结果该方法检测人源血液的相对荧光单位(RFU)值显著高于非血液(P<0.0001)以及非人源血液样本(P<0.0001),对血液RNA的检出灵敏度可达1×10^(-3) ng,且可成功检测出混合斑迹以及陈旧降解斑迹中的微量血液成分。结论本研究成功建立了一种基于CRISPR/Cas技术的SHERLOCK-HBA检测体系,能准确、灵敏地鉴定血液,操作简单、快速,为快速识别血液类样本提供了新的思路和参考。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 speciesspecific 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/Casbased 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 techniq

关 键 词：法医遗传学 血液鉴定 血红蛋白α CRISPR/Cas SHERLOCK 

分 类 号：DF795.2[医药卫生—法医学]