Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom

doi:10.12116/j.issn.1004-5619.2021.410801

Abstract

Abstract: Objective

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method.

Methods

The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles （New Plant genomic DNA extraction kit and Plant DNA Isolation kit） and one whole blood DNA extraction kit （whole blood genomic DNA extraction kit） were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed.

Results

When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant （P<0.05）. The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant （P<0.05）. However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group （P<0.05）.

Conclusion

The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Key words: forensic pathology, drowning, DNA extraction, diatom, glass bead-vortex oscillation improving method

CLC Number:

DF795.1

Jie CAI, Bo WANG, Sun-lin HU, Yi-hong QU, Tao SONG, Jian-hua CHEN, Jian-qiang DENG. Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom[J]. Journal of Forensic Medicine, 2022, 38(1): 119-126.

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URL: http://www.fyxzz.cn/EN/10.12116/j.issn.1004-5619.2021.410801

http://www.fyxzz.cn/EN/Y2022/V38/I1/119

Figures/Tables 5

Tab. 1 Combination of different glass bead proportion and vortex oscillation time

振荡时间/min	大小玻璃珠混合质量比
振荡时间/min	1∶2	1∶1	2∶1
2	①	②	③
3	④	⑤	⑥
4	⑦	⑧	⑨
5	⑩	?	?
6	?	?	?

Fig. 1 The electrophoresis result of different glass bead mass proportions and vortex oscillation time combinations

Fig. 2 Electrophoresis results of PCR amplification products extracted from three kits extracts

Fig. 3 Electrophoresis results of PCR amplification products extracted from four kits

Tab. 2 Comparison of Ct values of water samples and tissues before and after improvement with three kits

试剂盒	水样		组织
试剂盒	常规组	改良组	常规组	改良组
PowerSoil	24.06±0.91	-	16.97±0.60^1）	-
TIANGEN	25.71±0.63^2）	24.33±0.58^3）	26.56±0.30^2）	20.97±1.38^2）3）
FOREGENE	22.16±0.32^2）	21.86±0.32^2）	25.98±1.26^2）	19.60±0.89^2）3）
BioTeKe	23.67±1.49	21.94±0.40^2）	26.56±1.07^2）	19.38±0.77^2）3）

References 25

1	STEPHENSON L， HEUVEL C VAN DEN， BYARD R W. The persistent problem of drowning - A difficult diagnosis with inconclusive tests[J]. J Forensic Leg Med，2019，66：79-85. doi：10.1016/j.jflm.2019.06.003 .
2	赵建，罗银洲，王玉仲，等. 肺组织硅藻定量分析在实验动物溺死诊断中的应用[J]. 法医学杂志，2019，35（6）：706-709. doi：10.12116/j.issn.1004-5619.2019.06.011 .
	ZHAO J， LUO Y Z， WANG Y Z， et al. Application of quantitative analysis of diatoms in lung tissue for the diagnosis of drowning of experimental animals[J]. Fayixue Zazhi，2019，35（6）：706-709.
3	LIN C Y， YEN W C， HSIEH H M， et al. Diatomological investigation in sphenoid sinus fluid and lung tissue from cases of suspected drowning[J]. Forensic Sci Int，2014，244：111-115. doi：10.1016/j.forsciint.2014.08.023 .
4	周玉倩. 硅藻18S rRNA鉴别实验家兔水中尸体死因的研究[D]. 芜湖：皖南医学院，2013.
	ZHOU Y Q. A study on detecting diatom 18S rRNA genes to identify cause of death by drow-ning on rabbits[D]. Wuhu： Wannan Medical College，2013.
5	丛斌. 法医病理学[M].5版. 北京：人民卫生出版社，2016.
	CONG B. Forensic pathology[M]. 5th ed. Beijing： People’s Medical Publishing House，2016.
6	ZHANG P， KANG X， ZHANG S， et al. The length and width of diatoms in drowning cases as the evidence of diatoms penetrating the alveoli-capillary barrier[J]. Int J Legal Med，2020，134（3）：1037-1042. doi：10.1007/s00414-019-02164-4 .
7	刘泉，陈轶群，柯技，等. 生前溺死与死后入水SD大鼠心脏与脾内硅藻检验的研究[J].系统医学，2018，3（11）：13-17. doi：10.19368/j.cnki.2096-1782.2018.11.013 .
	LIU Q， CHEN Y Q， KE J， et al. Research on test of heart and diatom in spleen of SD rats drowning before death and entering the water after death[J]. Xitong Yixue，2018，3（11）：13-17.
8	付志鑫. 长白山地区桥弯藻科（Cymbellaceae）和舟形藻科（Naviculaceae）植物的分类学研究[D].哈尔滨：哈尔滨师范大学，2018.
	FU Z X. Taxonomical study of Cymbellaceae and Naviculaceae （bacillariophttad） from Changbai mountains Region. China[D]. Harbin： Harbin Normal University，2018.
9	胡鸿钧，魏印心. 中国淡水藻类：系统、分类及生态[M].北京：科学出版社，2006.
	HU H J， WEI Y X. The freshwater algae of China： Systematics， taxonomy and ecology[M]. Beijing： Science Press，2006.
10	YU Z H， XU Q Y， XIAO C， et al. SYBR Green real-time qPCR method： Diagnose drowning more rapidly and accurately[J]. Forensic Sci Int，2021，321：110720. doi：10.1016/j.forsciint.2021.110720 .
11	贺玉晓，郑永坤，李卫国，等. 丹江口水库早春真核浮游植物群落结构特征及其与环境因子的关系[J]. 环境科学学报，2021，41（6）：2192-2200. doi：10.13671/j.hjkxxb.2020.0550 .
	HE Y X， ZHENG Y K， LI W G， et al. Characteristics of eukaryotic phytoplankton community structure in early spring and its relationship with environmental factors in Danjiangkou Reservoir[J]. Huanjing Kexue Xuebao，2021，41（6）：2192-2200.
12	梁文贤，黄福墩，崔朋辉，等. 森林土壤微生物总DNA的提取方法[J]. 福建林学院学报，2014，34（2）：126-130. doi：10.13324/j.cnki.jfcf.2014.02.006 .
	LIANG W X， HUANG F D， CUI P H， et al. Extraction of total microbial DNA from forest soil[J]. Fujian Linxueyuan Xuebao，2014，34（2）：126-130.
13	王志龙，李春筱，蔡一鸣，等. 不同DNA提取方法对高通量测序检测沉积物微生物多样性的影响[J]. 昆明理工大学学报（自然科学版），2021，46（5）：89-99. doi：10.16112/j.cnki.53-1223/n.2021.05.251 .
	WANG Z L， LI C X， CAI Y M， et al. Study on the effect of different DNA extraction methods on high-throughput sequencing to detect microbial diversity in sediments[J]. Kunming Ligong Daxue Xuebao （Natural sciences），2021，46（5）：89-99.
14	THORNTON I， CARTOZZO C， MAYS D， et al. Development of a quantitative PCR-based method for studying temporal DNA degradation in waterlogged bone[J]. J Forensic Sci，2021，66（3）：1114-1123. doi：10.1111/1556-4029.14641 .
15	YUAN J, LI M Z, LIN S J. An improved DNA extraction method for efficient and quantitative recovery of phytoplankton diversity in natural assemblages[J]. PLoS One，2015，10（7）：e0133060. doi：10.1371/journal.pone.0133060 .
16	天根生化科技（北京）有限公司. 多糖多酚植物基因组DNA提取试剂盒说明书[EB/OL]. （2021-09-15）[2022-02-26]. .
	Biotech（Beijing）Co. Tiangen， Ltd. Super Plant Genomic DNA Kit （Polysacchardes & Polyphenolics-rich） instruction manual[EB/OL]. （2021-09-15）[2022-02-26]. .
17	成都福际生物技术有限公司. Plant DNA Isolation试剂盒说明书[EB/OL]. （2021-07-01）[2022-02-26]. .
	Foregene Co., Ltd.. FOREGENE Plant DNA Isolation kit instruction manual[EB/OL]. （2021-07-01）[2022-02-26]. .
18	ZIMMERMANN J， JAHN R， GEMEINHOLZER B. Barcoding diatoms： Evaluation of the V4 subregion on the 18S rRNA gene， including new primers and protocols[J]. Org Divers Evol，2011，11（3）：173-192. doi：10.1007/s13127-011-0050-6 .
19	李金明. 实时荧光PCR技术[M]. 2版. 北京：科学出版社，2016.
	LI J M. Real-time PCR[M]. 2nd ed. Beijing： Science Press，2016.
20	胡荣飞，陈璐瑶，张小妮，等. 利用研磨仪快速提取基因组DNA用于PCR[J]. 生物技术通讯，2019，30（5）：668-673. doi：10.3969/j.issn.1009-0002.2019.05.014 .
	HU R F， CHEN L Y， ZHANG X N， et al. Rapid extraction genomic DNA for PCR by grin-ding mill[J]. Shengwu Jishu Tongxun，2019，30（5）：668-673.
21	刘向东，刘超，徐曲毅，等. 实时荧光定量PCR检测硅藻UPA基因在溺死诊断中的研究[J]. 中国法医学杂志，2018，33（2）：124-129. doi：10.13618/j.issn.1001-5728.2018.02.003 .
	LIU X D， LIU C， XU Q Y， et al. A real-time qPCR method to identify diatom UPA gene for the drowning diagnosis[J]. Zhongguo Fayixue Zazhi，2018，33（2）：124-129.
22	KOBAYASHI M， YAMADA Y， ZHANG W D， et al. Novel detection of plankton from lung tissue by enzymatic digestion method[J]. Forensic Sci Int，1993，60（1/2）：81-90. doi：10.1016/0379-0738（93）90095-R .
23	黄罗冬，高保燕，王飞飞，等. 真核藻类基因组研究进展[J]. 基因组学与应用生物学，2020，39（8）：3626-3646. doi：10.13417/j.gab.039.003626 .
	HUANG L D， GAO B Y， WANG F F， et al. Research advances in the genome of eukaryotic algae[J]. Jiyinzuxue Yu Yingyong Shengwuxue，2020，39（8）：3626-3646.
24	张平萍，刘蕴泽，康晓东，等. 溺死尸体组织与溺液中硅藻种类的一致性研究[J]. 中国法医学杂志，2020，35（4）：394-397，402. doi：10.13618/j.issn.1001-5728.2020.04.012 .
	ZHANG P P， LIU Y Z， KANG X D， et al. Study on the concordance of diatom patterns in organs and drowning medium[J]. Zhongguo Fayixue Zazhi，2020，35（4）：394-397，402.
25	李欢，余仲昊，刘超. 高通量测序技术在浮游生物研究中的应用[J]. 广东公安科技，2020，28（4）：57-58.
	LI H， YU Z H， LIU C. Application of high throughput sequencing technology in plankton research[J]. Guangdong Gongan Keji，2020，28（4）：57-58.

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