血液鑑別方法及DNA鑑定系統靈敏度之評估

Evaluation of the Blood Identification Methods and Sensitivity of DNA Identification Systems

10.6134/tjfm.2013.0501.03

謝幸媚(Hsing-Mei Hsieh)；盧皇德(Huang-Te Lu)；楊雅玲(Ya-Ling Yang)；蔡麗琴(Li-Chin Tsai)

體液鑑定 ； 血液鑑定 ； 血液初步試驗 ； 血液確認試驗 ； DNA鑑定 ； Body fluid identification ； Blood identification ； Blood preliminary test ； Blood confirmatory test ； DNA identification

台灣法醫學誌

5卷1期（2013 / 06 / 01）

28 - 40

繁體中文

本研究以TMB、KM、Luminol及BlueStar試驗進行血液初篩方法的評估，TMB、KM及BlueStar試驗只須0.31～3.125 nl液態血液即可檢出其陽性反應；血斑則以TMB較佳；血斑於不同溫度（-20℃、4℃、室內常溫、室外常溫及40℃）儲存1年以上仍可以TMB、Luminol及BlueStar 試驗檢出其陽性反應；4種血斑初篩試劑於-20℃儲存1年以上仍具有檢出能力；特異性評估之結果發現除了TMB試驗會對其他8種生物性檢體產生偽陽性反應外，另3種方法均未發現偽陽性反應。以Seratec HemDirect Hemoglobin Assay、ABAcard HemaTrace Test及RSID-Blood Test等三種免疫層析方法進行確認試驗之評估，發現Seratec HemDirect Hemoglobin Assay及ABAcard HemaTrace Test於液態血液及血斑之靈敏度較佳，約介於1.5 nl至3.75 nl之間；RSID-Blood Test則介於25 nl至100 nl之間；特異性試驗之結果顯示除了ABAcard HemaTrace Test外其他兩個方法均不會產生陽性反應。DNA鑑定系統靈敏度評估之結果，發現微量血液或血斑所萃取之DNA只需500 pg以上即可得到細胞核STR基因型或Y-STR單倍型之完整圖譜；以62.5 pg以上之DNA當模板即可獲得D-loop序列明顯的PCR產物。本研究成果可提供刑事實驗室進行疑似血液檢體初步及確認試驗方法選擇之參考，以達到快速篩檢的目的，並提升DNA鑑驗之效能。

TMB, KM, Luminol and BlueStar tests were used to evaluate the blood preliminary tests in this study. It only needs 0.31 ~ 25 nl liquid blood to have the positive result for the reagents of TMB, KM and BlueStar. TMB showed higher sensitivity than the other three methods for the blood stains. The positive results for TMB, Luminol and BlueStar tests were shown when the blood stains were stored under five different conditions (-20 ℃, 4 ℃, indoor temperature, outdoor temperature and 40 ℃) for more than one year. These four reagents for blood preliminary test were still effective while stored at -20 ℃ for more than one year. For the specificity test of these four methods, other eight biological specimens showed the pseudo-positive results for TMB test, and no any pseudo-positive results was found for the other three methods. Three Immunochromatographic membrane test (Seratec HemDirect Hemoglobin assay, ABAcard HemaTrace test and RSID-Blood test) were used to evaluate the confirmatory tests for liquid blood and blood stain. Seratec HemDirect Hemoglobin assay and ABAcard HemaTrace test showed higher sensitivity than RSID-Blood test. It needs 1.5 nl to 3.75 nl blood to obtain the positive result for Seratec HemDirect Hemoglobin assay and ABAcard HemaTrace test, but 25 nl to 100 nl for RSID-Blood test. For the specificity test, there were no pseudo-positive results for Seratec HemDirect Hemoglobin assay and RSID-Blood test but not for ABAcard HemaTrace test. For the sensitivity evaluation of nuclear STR and Y-STR DNA identification systems, the full profile could be obtained when 500 pg DNA from liquid blood or blood stain were inputted. For mitochondrial D-loop amplification, 62.5 pg of input DNA was enough. The results of this study could provide the reference for the forensic laboratory to choose the blood preliminary test and confirmatory test while performing the test for suspected blood specimens. It will reach the purposes of rapid screen and improving the efficiency of DNA identification.

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