Rapid On-Site Detection of Male DNA Using Direct Fluorescence-Based Recombinase Polymerase Amplification (RPA)

국문 초록 (Abstract)

범죄 현장에서 남성 유래 DNA를 신속하게 식별하는 것은 유효 증거물의 선별과 초기 수사 방향 설정에 결정적인 역할을 한다. 본 연구의 목적은 남성 특이적 SRY 유전자를 표적으로 하는 형광 기반 Recombinase Polymerase Amplification (RPA) 분석법을 개발·최적화하고, 이를 현장에서 적용 가능한 신속한 남성 DNA 검출 기법으로 평가하는 것이다.

SYBR Green I 형광 검출을 기반으로 반응 부피, 증폭 온도, 반응 시간, 프라이머 농도 등 주요 RPA 조건을 체계적으로 최적화하였으며, Limit of Blank (LoB)· Limit of Detection (LoD) 분석을 통해 형광 판독 기준을 확립하였다. 또한 증류수(Distilled water)와 100 mM NaOH를 이용한 직접 용해법(Direct lysis)를 전혈과 혈흔 스왑에 적용하여 현장 간소화 절차의 가능성을 검증하고, 손바닥·팔 안쪽·겨드랑이를 활용한 체온 기반 인큐베이션 조건을 평가함으로써 장비 비의존적 증폭 가능성을 확인하였다. 모의 범죄현장 조건을 반영하기 위해 담배꽁초 및 음료 용기 시료를 분석하였으며, 비교를 위해 동일 표적에 대한 비색 Loop-mediated isothermal amplification (LAMP) 분석을 수행하였다.

최적화 결과, RPA는 25 µL, 37 °C, 20분, 프라이머 0.48 µM 조건에서 명확한 형광 구분을 보였고, 검출한계는 100 pg으로 확인되었다. 증폭은 남성 유래 혈액·타액에서만 관찰되어 높은 특이도를 보였으며, NaOH 기반 직접 용해법은 증류수보다 강하고 일관된 증폭 효율을 나타냈다. 체온 기반 인큐베이션 조건에서도 안정적인 증폭이 이루어졌고, 모의 증거물 분석에서도 남성 시료에서만 양성 결과가 확인되었다. 비색 LAMP는 증류수 기반 직접 용해법에서는 안정적으로 증폭되었으나 NaOH 조건에서는 호환성이 제한되었다. 반면, RPA는 두 조건 모두에서 일관된 증폭을 보여 다양한 직접 용해법 조건과 현장 환경에 보다 유연하게 적용될 수 있는 것으로 판단된다.

종합적으로, 본 연구에서 개발된 형광 기반 RPA 분석법은 신속성·민감도·간편성을 모두 갖춘 남성 DNA 현장 스크리닝 기법으로서 높은 적용 가능성을 보였다. 향후 휴대형 시스템과의 통합을 통해 범죄현장에서의 유효 증거물 선별 및 초기 수사 의사결정에 기여할 것으로 기대된다.

다국어 초록 (Multilingual Abstract)

Rapid detection of male-derived DNA at crime scenes is essential for efficient evidence screening and early investigative decisions. This study developed and optimized a fluorescence-based recombinase polymerase amplification (RPA) assay targeting the male-specific SRY gene and evaluated its suitability as a rapid, equipment-minimal method for on-site male DNA detection.

An SYBR Green I fluorescence detection system was incorporated into the RPA workflow, and reaction conditions were optimized by examining reaction volume, amplification temperature, amplification time, and primer concentration. Fluorescence thresholds were established through Limit of Blank (LoB) and Limit of Detection (LoD) analyses. Field applicability was assessed using distilled water and 100 mM NaOH direct lysis applied to both whole blood and dried blood swabs, as well as body heat–based incubation at three anatomical sites. Forensic mock samples, including cigarette butts and beverage containers, were also tested alongside a colorimetric LAMP assay targeting the same locus.

Under the optimized conditions (25 µL, 37 °C, 20 min, 0.48 µM primers), the RPA assay showed clear fluorescence discrimination with a detection limit of 100 pg and demonstrated high specificity, amplifying only male-derived blood and saliva. NaOH-based direct lysis produced stronger and more consistent amplification than distilled water, and body heat–based incubation supported stable amplification. Mock samples yielded positive results only for male-derived items. Although colorimetric LAMP showed comparable sensitivity and functioned under distilled water–based lysis, its compatibility with NaOH was limited, whereas RPA consistently performed under both lysis conditions, indicating greater flexibility for simplified field workflows.

Overall, the optimized fluorescence-based RPA assay provides a rapid, sensitive, and practical approach for on-site male DNA screening. Its compatibility with simplified lysis and the feasibility of body heat–based incubation highlight its strong potential as a point-of-crime-scene tool that supports effective evidence selection and early investigative decisions.

목차 (Table of Contents)

• Chapter 1. Introduction 1
• 1.1. Need for rapid screening of male-derived DNA in forensic investigations 1
• 1.2. Male-specific genetic marker: SRY 4
• 1.3. Isothermal amplification techniques 6
• 1.3.1. Loop-mediated isothermal amplification (LAMP) 8
• 1.3.2. Recombinase polymerase amplification (RPA) 11
• 1.4. Detection principles of RPA and LAMP 15
• 1.5. Purpose of the study 18
• Chapter 2. Materials and Methods 20
• 2.1. Biological sample preparation 22
• 2.1.1. Specimen collection and ethical approval 22
• 2.1.2. DNA extraction 23
• 2.1.3. DNA quantification 24
• 2.2. Direct lysis method using biological sample 26
• 2.2.1. Distilled water 26
• 2.2.2. NaOH solution 27
• 2.3. Development of fluorescence-based RPA assay 29
• 2.3.1. Design of RPA primers 29
• 2.3.2. Standard RPA reaction protocol 31
• 2.3.3. Fluorescence detection 33
• 2.3.4. Fluorescence intensity analysis 35
• 2.3.5. Establishment of thresholds 36
• 2.3.6. Agarose gel electrophoresis 37
• 2.4. Optimization of RPA protocol 38
• 2.4.1. Reaction volume 38
• 2.4.2. Amplification temperature 41
• 2.4.3. Amplification time 42
• 2.4.4. Primer concentration 43
• 2.5. Validation of RPA assay 44
• 2.5.1. Sensitivity test 44
• 2.5.2. Specificity test 45
• 2.6. Evaluation of on-site applicability 46
• 2.6.1. Body-heat based assay 46
• 2.6.2. Application of RPA assay to forensic mock samples 48
• 2.7. Colorimetric LAMP assay for comparative evaluation 50
• Chapter 3. Results and Discussion 56
• 3.1. Optimization of RPA protocol 56
• 3.1.1. Reaction volume 56
• 3.1.2. Amplification temperature 58
• 3.1.3. Amplification time 60
• 3.1.4. Primer concentration 62
• 3.2. Validation of RPA assay 64
• 3.2.1. Sensitivity test 64
• 3.2.2. Specificity test 66
• 3.3. Direct lysis method using biological sample 69
• 3.3.1. Distilled water 69
• 3.3.2. NaOH solution 70
• 3.4. Evaluation of on-site applicability 73
• 3.4.1. Body-heat based assay 73
• 3.4.2. Application of RPA assay to forensic mock samples 75
• 3.5. Colorimetric LAMP assay for comparative evaluation 76
• 3.5.1. Optimization of LAMP protocol 76
• 3.5.2. Validation of LAMP assay 78
• 3.5.3. Direct lysis method using biological sample 82
• 3.5.4. Application of LAMP assay to forensic mock samples 85
• 3.6. Comparison of RPA and LAMP assays 86
• Chapter 4. Conclusion 89
• Reference 93
• 국문 초록 97