{"title":"土壤中细胞外DNA提取试剂盒的评价","authors":"H. Miyaguchi, Hiroaki Nakahara, R. Sugita","doi":"10.3408/JAFST.748","DOIUrl":null,"url":null,"abstract":"Five commercial DNA extraction kits for microbial DNA in soil were evaluated in terms of the extraction e‹ciencies of artiˆcially-spiked extracellular DNA in soil. Commercially-available puriˆed DNA derived from the sperm of Clupea harengus (Atlantic herring) was homogeneously premixed in blank soil samples and extracted using the kits. The spiked DNA in each of the extracts was quantitated by real-time PCR to evaluate extraction e‹ciency using a speciˆc primer set for the mitochondrial 16S rRNA gene of Clupea harengus. Only the Extrap Soil DNA Kit could extract the DNA from all types of soils spiked at 10 mg/g soil (vegetable garden soil 0.85, courtyard soil 0.30 and virgin andosol 0.027), whereas other kits (Nucleospin Soil, Power Soil DNA Isolation Kit, ISOIL and ISOIL for Beads Beating) could not extract the DNA larger than the quantitation limit (0.020) from the courtyard soil and the virgin andosol which exhibited high phosphate absorption coe‹cient. Skim milk concentration and bead-beating time using the Extrap Soil DNA Kit were optimized for 20 mg/g soil and 30 s, respectively. Signiˆcant PCR inhibition was not observed under the optimized condition.","PeriodicalId":14709,"journal":{"name":"Japanese Journal of Forensic Science and Technology","volume":"36 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"3","resultStr":"{\"title\":\"Evaluation of the commercial kits for the extraction of extracellular DNA in soil\",\"authors\":\"H. Miyaguchi, Hiroaki Nakahara, R. Sugita\",\"doi\":\"10.3408/JAFST.748\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Five commercial DNA extraction kits for microbial DNA in soil were evaluated in terms of the extraction e‹ciencies of artiˆcially-spiked extracellular DNA in soil. Commercially-available puriˆed DNA derived from the sperm of Clupea harengus (Atlantic herring) was homogeneously premixed in blank soil samples and extracted using the kits. The spiked DNA in each of the extracts was quantitated by real-time PCR to evaluate extraction e‹ciency using a speciˆc primer set for the mitochondrial 16S rRNA gene of Clupea harengus. Only the Extrap Soil DNA Kit could extract the DNA from all types of soils spiked at 10 mg/g soil (vegetable garden soil 0.85, courtyard soil 0.30 and virgin andosol 0.027), whereas other kits (Nucleospin Soil, Power Soil DNA Isolation Kit, ISOIL and ISOIL for Beads Beating) could not extract the DNA larger than the quantitation limit (0.020) from the courtyard soil and the virgin andosol which exhibited high phosphate absorption coe‹cient. Skim milk concentration and bead-beating time using the Extrap Soil DNA Kit were optimized for 20 mg/g soil and 30 s, respectively. Signiˆcant PCR inhibition was not observed under the optimized condition.\",\"PeriodicalId\":14709,\"journal\":{\"name\":\"Japanese Journal of Forensic Science and Technology\",\"volume\":\"36 1\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"3\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Japanese Journal of Forensic Science and Technology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3408/JAFST.748\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Japanese Journal of Forensic Science and Technology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3408/JAFST.748","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 3
摘要
对5种市售土壤微生物DNA提取试剂盒对土壤中人工加钉细胞外DNA的提取效果进行了评价。从大西洋鲱鱼(Clupea harengus)精子中提取的市售纯化DNA在空白土壤样品中均匀混合,并使用试剂盒提取。采用实时荧光定量PCR对各提取液中的加标DNA进行定量分析,以评价提取效果,采用的引物为Clupea harengus线粒体16S rRNA基因特异性引物。在10 mg/g土壤中,只有Extrap土壤DNA试剂盒能从所有类型的土壤(菜园土壤0.85、庭院土壤0.30、原生土0.027)中提取DNA,而其他试剂盒(Nucleospin土壤、Power土壤DNA分离试剂盒、ISOIL和ISOIL for Beads Beating)均不能从庭院土壤和具有高磷酸盐吸收率的原生土中提取出大于定量限(0.020)的DNA。使用Extrap Soil DNA Kit优化脱脂奶浓度和打珠时间,分别为20 mg/g土壤和30 s。在优化条件下,未观察到明显的PCR抑制作用。
Evaluation of the commercial kits for the extraction of extracellular DNA in soil
Five commercial DNA extraction kits for microbial DNA in soil were evaluated in terms of the extraction e‹ciencies of artiˆcially-spiked extracellular DNA in soil. Commercially-available puriˆed DNA derived from the sperm of Clupea harengus (Atlantic herring) was homogeneously premixed in blank soil samples and extracted using the kits. The spiked DNA in each of the extracts was quantitated by real-time PCR to evaluate extraction e‹ciency using a speciˆc primer set for the mitochondrial 16S rRNA gene of Clupea harengus. Only the Extrap Soil DNA Kit could extract the DNA from all types of soils spiked at 10 mg/g soil (vegetable garden soil 0.85, courtyard soil 0.30 and virgin andosol 0.027), whereas other kits (Nucleospin Soil, Power Soil DNA Isolation Kit, ISOIL and ISOIL for Beads Beating) could not extract the DNA larger than the quantitation limit (0.020) from the courtyard soil and the virgin andosol which exhibited high phosphate absorption coe‹cient. Skim milk concentration and bead-beating time using the Extrap Soil DNA Kit were optimized for 20 mg/g soil and 30 s, respectively. Signiˆcant PCR inhibition was not observed under the optimized condition.