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⁃PC...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。展开更多
Background: A deletion of 32 bp in the nucleotide sequence of CCR5 gene results in a defective CCR5 which confers protection from HIV infection in the homozygous state, while reducing the rate of disease progression t...Background: A deletion of 32 bp in the nucleotide sequence of CCR5 gene results in a defective CCR5 which confers protection from HIV infection in the homozygous state, while reducing the rate of disease progression to AIDS and death in the heterozygous state. The status of the CCR5Δ32 gene has not been reported in Nigeria. Aim: This study was aimed at analyzing single nucleotide polymorphism of CCR5 gene among the Ijaws resident in Yenagoa, Nigeria. Methods: 100 subjects (75 HIV negative and 25 HIV positive control) were recruited for this study. The CCR5 genes were amplified by 2 Stage PCR reaction using GeneAmp 9700 PCR system utilizing specific primers that would flank 32 bp deletion, followed by agarose gel electrophoresis, DNA sequencing of 20 subjects was done followed by phylogenetic and polymorphism analysis. Results: The results showed that 75 (100%) of the HIV negative subjects had 189 base pair in their CCR5 gene. Nucleotide of the 20 (100%) of the sequenced samples were conservatively same and no SNP was observed. Conclusion: This study documented no SNPs in CCR5 gene of the study population hence;the study population has no protection from HIV infection.展开更多
文摘建立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。
文摘Background: A deletion of 32 bp in the nucleotide sequence of CCR5 gene results in a defective CCR5 which confers protection from HIV infection in the homozygous state, while reducing the rate of disease progression to AIDS and death in the heterozygous state. The status of the CCR5Δ32 gene has not been reported in Nigeria. Aim: This study was aimed at analyzing single nucleotide polymorphism of CCR5 gene among the Ijaws resident in Yenagoa, Nigeria. Methods: 100 subjects (75 HIV negative and 25 HIV positive control) were recruited for this study. The CCR5 genes were amplified by 2 Stage PCR reaction using GeneAmp 9700 PCR system utilizing specific primers that would flank 32 bp deletion, followed by agarose gel electrophoresis, DNA sequencing of 20 subjects was done followed by phylogenetic and polymorphism analysis. Results: The results showed that 75 (100%) of the HIV negative subjects had 189 base pair in their CCR5 gene. Nucleotide of the 20 (100%) of the sequenced samples were conservatively same and no SNP was observed. Conclusion: This study documented no SNPs in CCR5 gene of the study population hence;the study population has no protection from HIV infection.
