基于多重RT⁃PCR扩增与双链cDNA合成的A组轮状病毒全基因组测序效果比较  

Comparison of Whole Genome Sequencing Effects of Group A Rotavirus Based on Multiplex RT⁃PCR Amplification and Double Stranded cDNA Synthesis

作　　者：黄枝妙 吴冰珊[1] 林琦[1] 林卫东 翁育伟[1] HUANG Zhimiao;WU Bingshan;LIN Qi;LIN Weidong;WENG Yuwei(Fujian Provincial Center for Disease Control and Prevention,Fujian Provincial Key Laboratory of Zoonosis Research,Fuzhou 350012,China;Fuzhou Pediatric Hospital,Fuzhou 350004,China)

机构地区：[1]福建省疾病预防控制中心福建省人兽共患病研究重点实验室,福州350012 [2]福州市儿童医院,福州350004

出　　处：《病毒学报》2024年第4期716-724,共9页Chinese Journal of Virology

基　　金：福建省卫健委青年科研课题(项目号:2021QNA035);题目:优化的多重RT⁃PCR扩增在轮状病毒临床标本二代测序中的应用。

摘　　要：建立A组轮状病毒(Group A rotavirus,RVA)全基因组单管多重RT⁃PCR扩增方法,并比较其与双链cDNA合成方法在RVA临床标本全基因组二代测序中的应用。对2022年某哨点医院的18份腹泻住院患儿RVA阳性粪便标本进行核酸提取,分别用单管多重RT⁃PCR扩增方法对RVA全基因组进行扩增,用随机引物对病毒核酸进行反转录及双链cDNA合成,最后采用Illumina平台对这2种不同类型的产物进行二代测序。用CLC软件对测序数据进行拼接,分析有效数据量、测序深度及全基因组覆盖度等参数,并用IGV软件查看全基因组测序深度覆盖轨迹。多重RT⁃PCR扩增产物测序和双链cDNA测序获得的RVA有效数据量分别为69.39%~99.83%和0.38%~92.99%,平均测序深度分别为10172×~68156×和35×~61783×,全基因组测序深度10×以上的位点覆盖度分别为98.93%~100.00%和86.28%~100.00%。从全基因组测序深度情况来看,多重RT⁃PCR扩增产物测序在同一个基因节段内部测序深度较为均匀,在11个节段之间测序深度差别较大;而双链cDNA测序在不同基因节段之间的测序深度较为均匀,但同一个基因节段内部靠近5'和3'末端测序深度较低。2种方法所获得的病毒核苷酸序列基本一致,部分基因节段在5'或3'末端100 bp区域内存在2个以内的核苷酸位点差异。在多重RT⁃PCR扩增方法中鉴于引物在不同基因型别间的特异性,对本研究中未涉及到的基因型别的扩增效率还有待进一步验证。单管多重RT⁃PCR扩增方法和双链cDNA合成方法虽然各有优缺点,但均适用于轮状病毒全基因组二代测序在基层的推广。To establish a single tube multiplex reverse transcription⁃polymerase chain reaction(RT⁃PCR)amplification method that targeted the whole genome of Group A rotavirus(RVA),and to compare the effect with the double stranded cDNA synthesis method applying in the next generation sequencing of RVA clinical specimens,nucleic acid was extracted from 18 RVA⁃positive fecal samples of hospitalized children with diarrhea from a monitoring hospital in 2022.The whole genome of RVA was amplified using a single tube multiplex RT⁃PCR method,and the double stranded cDNA synthesized after the RNA was reverse transcripted with random primers.The Illumina platform was used for next generation sequencing of these two different types of products.CLC software was used to map the sequencing data,and analyze the effective data amount,sequencing depth,and whole genome coverage.The IGV software was used to view the sequencing coverage trace throughout the whole genome.The effective data amount of multiplex RT⁃PCR amplification products sequencing and double⁃stranded cDNA sequencing were 69.39%~99.83%and 0.38%~92.99%,respectively.The average sequencing depth was 10172×~68156×and 35×~61783×,respectively,and the whole genome coverage of sequencing depth of more than 10×was 98.93%~100%and 86.28%~100%,respectively.From the perspective of whole genome sequencing depth,the multiplex RT⁃PCR amplification products sequencing was relatively uniform in the depth of the same gene segment and quite different among the 11 segments.However,the double⁃stranded cDNA sequencing showed a uniform depth between different gene segments,but the sequencing depth near the 5'and 3'ends of the same segment was lower.The nucleotide sequences obtained by both methods were basically consistent,some gene segments showed 1~2 different nucleotide sites within the 100bp region near the 5'or 3'end.Due to the specificity of primers among different genotypes,the multiplex RT⁃PCR efficiency need to be further verified for rotavirus types that was not cove

关 键 词：A组轮状病毒 全基因组测序 多重RT⁃PCR扩增 扩增子测序 双链cDNA合成 

分 类 号：R373.2[医药卫生—病原生物学]