摘要
目的优化和评估粪便和组织样本高通量测序前处理方法,提高高通量测序在宏病毒组研究中的灵敏度。方法针对粪便样本,选取经粪便排毒的5种病毒阳性样本混合模拟样本,对不同材质的滤器、核酸酶不同处理时间、不同的提取核酸方法进行评价。针对组织样本,选取动物组织中检出的2种病毒阳性样本,对过滤、核酸酶处理和不同的核酸提取方法进行评价。采用TaqMan real-time PCR定量方法评价每步处理对每一种模式病毒核酸载量的影响,采用SYBR Green real-time PCR方法对细菌16S rRNA基因和宿主12S rRNA基因进行定量以评价细菌和宿主的去除效果。结果对于粪便样本,使用0.22μm的PES滤器过滤效果较好,使用PVDF材质滤器导致样本量减少;核酸酶消化2 h比消化1 h去除细菌的效果更好,且病毒损失较少;使用RPMK试剂盒可以有效减少细菌,但对部分病毒提取效果较差,而MVSK试剂盒提取病毒核酸效果较好。对于组织样本,使用0.22μm的PES滤器过滤病毒损失较少;核酸酶消化1 h可以有效去除宿主gDNA,且病毒损失较少;使用VNAEK II试剂盒提取病毒核酸效果最好,Trizol LS+RPMK方法去除宿主gDNA效果较好,但病毒损失较大。粪便和组织样本的前处理方法整个流程的病毒损失分别为(1.7-3.0)Ct和(1.6-2.5)Ct。结论粪便样本最佳方法为PES滤器过滤、核酸酶消化2 h、MVSK试剂盒提取核酸;组织样本最佳方法为PES滤器过滤、核酸酶消化1 h、VNAEK II试剂盒提取核酸。
Objective To optimize and evaluate the pretreatment method for high-throughput sequencing of fecal and tissue samples to improve the sensitivity of high-throughput sequencing in the study of virome.Methods For fecal samples,five virus-positive samples that have been detoxified from feces were selected,mixed as the simulated samples,and filters made of different materials were used,different processing times were set for nucleases,and different kits were used to extract nucleic acid.For tissue samples,two virus-positive samples that have been detected in animal tissues,filter and nuclease treatment were used,and different extraction method were used to extract nucleic acid.TaqMan real-time PCR was used to quantitatively evaluate the impact of each treatment on the virus and the advantages and disadvantages were compared.We used the SYBR Green real-time PCR quantitative method to evaluate the removal effect of the above method on bacteria 16S rRNA and host 12S rRNA genome.Results For fecal samples,the 0.22μm PES filter showed a better filtering effect,and the PVDF material filter reduces the sample volume;2 h nuclease digestion was better than 1 h digestion to remove bacteria,and the virus loss was less;the use of RPMK kits can effectively reduce bacteria,but the effect of extracting some viruses was poor,and the MVSK kit has a better effect of extracting viral nucleic acid.For tissue samples,0.22μm PES filter filtration,nuclease digestion for 1 h and VNAEK II kit extraction of nucleic acid were the best,Trizol LS combined with the RPMK method was better for gDNA removal,but the virus loss was larger.The virus loss of the whole process of the pretreatment method of feces and tissue samples was(1.7-3.0)Ct and(1.6-2.5)Ct,respectively.Conclusions The optimal method for fecal samples was to filter with a PES filter,then digest with nuclease for 2 hours,and then extract nucleic acids using the MVSK kit;the optimal method for tissue samples was to filter with a PES filter,then perform 1 h nuclease digestion,and then us
作者
马晓华
柴萨萨
李利利
郑贵森
段招军
Ma Xiaohua;Chai Sasa;Li Lili;Zheng Guisen;Duan Zhaojun(School of Public Health,Gansu University of Chinese Medicine,Lanzhou 730000,China;National Institute for Viral Disease Control and Prevention,China CDC,Beijing 100000,China;School of Basic Medicine,North China University of Science and Technology,Tangshan 063000,China)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2022年第1期103-110,共8页
Chinese Journal of Experimental and Clinical Virology
基金
国家科技重大专项(2018ZX10305409-004-002)。
关键词
高通量测序
病毒
前处理
方法学
粪便
组织
high-throughput sequencing
Virus
Pretreatment
Methodology
Feces
Tissue
