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Real-time polymerase chain reaction assay for vaginal CpG marker, Vag1 region, and trial for precise vaginal fluid identification using a supplemental CpG marker.

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Τίτλος: Real-time polymerase chain reaction assay for vaginal CpG marker, Vag1 region, and trial for precise vaginal fluid identification using a supplemental CpG marker.
Συγγραφείς: Watanabe K; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan. Electronic address: k-watanabe@nrips.go.jp., Shimura T; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan; Forensic Science Laboratory, Tokyo Metropolitan Police Department, 3-35-21 Shakujiidai, Nerima-ku, Tokyo, Japan., Yamagishi T; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan., Toyomane K; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan., Iwase H; Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba, Chiba 260-8670, Japan., Akutsu T; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
Πηγή: Legal medicine (Tokyo, Japan) [Leg Med (Tokyo)] 2026 Sep; Vol. 85, pp. 102946. Date of Electronic Publication: 2026 Jul 28.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: Elsevier Science Ireland Ltd Country of Publication: Ireland NLM ID: 100889186 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4162 (Electronic) Linking ISSN: 13446223 NLM ISO Abbreviation: Leg Med (Tokyo) Subsets: MEDLINE
Imprint Name(s): Publication: <2003->: East Park, Co. Clare, Ireland : Elsevier Science Ireland Ltd.
Original Publication: Tokyo : Japanese Society of Legal Medicine, c1999-
Ιατρικοί όροι (MeSH): Vagina*/metabolism , Real-Time Polymerase Chain Reaction*/methods , CpG Islands*/genetics , Body Fluids*/chemistry, Female ; Humans ; DNA Methylation ; Genetic Markers
Περίληψη: The identification of vaginal secretions in forensic samples is often required to reconstruct sexual assault events; however, few conventional confirmatory test exists for vaginal fluid identification. The Vag1 region (cg14991487) is a marker previously reported as vaginal-specific; therefore, we developed a fluorescent-probe-based real-time polymerase chain reaction (PCR) assay suitable for routine forensic analysis that simultaneously detects the methylation status of two adjacent CpG sites within the Vag1 region. The assay yielded a positive methylation signal for vaginal DNA, while showing no cross-reactivity with blood, semen, or saliva DNA; however, a subset of anorectal samples also produced a positive signal. Sensitivity testing showed reliable detection with as little as 0.5 ng of vaginal DNA, although increasing amounts of non-vaginal DNA attenuated the signal in mixed samples. In vaginal-semen mixtures, the semen component masked the signal, but differential extraction of the epithelial fraction restored detection. To address anorectal cross-reactivity, we screened the genome-wide methylation data and identified cg14541992 as an additional marker. The marker is almost completely demethylated in vaginal DNA and highly methylated in anorectal DNA. A duplex assay targeting Vag1 and cg14541992 was established, which could exclude anorectal samples from Vag1-positive samples, although false exclusions of vaginal samples can occur in mixtures (e.g., mixtures with anorectal, semen, or blood DNA). We believe that the rapidity and simplicity of the developed real-time PCR assay are practical advantages for forensic vaginal fluid identification.
(Copyright © 2026 Elsevier B.V. All rights reserved.)
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Contributed Indexing: Keywords: DNA methylation; Real-time polymerase chain reaction; Vaginal fluid
Substance Nomenclature: 0 (Genetic Markers)
Entry Date(s): Date Created: 20260730 Date Completed: 20260822 Latest Revision: 20260822
Update Code: 20260823
DOI: 10.1016/j.legalmed.2026.102946
PMID: 42531738
Βάση Δεδομένων: MEDLINE
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  Data: Real-time polymerase chain reaction assay for vaginal CpG marker, Vag1 region, and trial for precise vaginal fluid identification using a supplemental CpG marker.
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  Data: &lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Watanabe+K%22&quot;&gt;Watanabe K&lt;/searchLink&gt;; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan. Electronic address: k-watanabe@nrips.go.jp.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Shimura+T%22&quot;&gt;Shimura T&lt;/searchLink&gt;; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan; Forensic Science Laboratory, Tokyo Metropolitan Police Department, 3-35-21 Shakujiidai, Nerima-ku, Tokyo, Japan.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Yamagishi+T%22&quot;&gt;Yamagishi T&lt;/searchLink&gt;; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Toyomane+K%22&quot;&gt;Toyomane K&lt;/searchLink&gt;; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Iwase+H%22&quot;&gt;Iwase H&lt;/searchLink&gt;; Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba, Chiba 260-8670, Japan.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Akutsu+T%22&quot;&gt;Akutsu T&lt;/searchLink&gt;; First Department of Forensic Science, National Research Institute of Police Science, 6-3-1, Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
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  Data: The identification of vaginal secretions in forensic samples is often required to reconstruct sexual assault events; however, few conventional confirmatory test exists for vaginal fluid identification. The Vag1 region (cg14991487) is a marker previously reported as vaginal-specific; therefore, we developed a fluorescent-probe-based real-time polymerase chain reaction (PCR) assay suitable for routine forensic analysis that simultaneously detects the methylation status of two adjacent CpG sites within the Vag1 region. The assay yielded a positive methylation signal for vaginal DNA, while showing no cross-reactivity with blood, semen, or saliva DNA; however, a subset of anorectal samples also produced a positive signal. Sensitivity testing showed reliable detection with as little as 0.5&#160;ng of vaginal DNA, although increasing amounts of non-vaginal DNA attenuated the signal in mixed samples. In vaginal-semen mixtures, the semen component masked the signal, but differential extraction of the epithelial fraction restored detection. To address anorectal cross-reactivity, we screened the genome-wide methylation data and identified cg14541992 as an additional marker. The marker is almost completely demethylated in vaginal DNA and highly methylated in anorectal DNA. A duplex assay targeting Vag1 and cg14541992 was established, which could exclude anorectal samples from Vag1-positive samples, although false exclusions of vaginal samples can occur in mixtures (e.g., mixtures with anorectal, semen, or blood DNA). We believe that the rapidity and simplicity of the developed real-time PCR assay are practical advantages for forensic vaginal fluid identification.&lt;br /&gt; (Copyright &#169; 2026 Elsevier B.V. All rights reserved.)
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