We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treate...We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.展开更多
目的探讨微滴数字聚合酶链反应(ddPCR)在重症急性胰腺炎(SAP)合并疑似血流感染(BSI)病原学诊断中的价值。方法选取2022年7—9月某院重症医学科收治的SAP患者,在疑似BSI发作时同步采集静脉血进行ddPCR检测和血培养(BC)及药敏试验(AST),...目的探讨微滴数字聚合酶链反应(ddPCR)在重症急性胰腺炎(SAP)合并疑似血流感染(BSI)病原学诊断中的价值。方法选取2022年7—9月某院重症医学科收治的SAP患者,在疑似BSI发作时同步采集静脉血进行ddPCR检测和血培养(BC)及药敏试验(AST),记录两种检测方法的耗时,比较ddPCR与BC的检测结果,计算ddPCR的病原学诊断效能,并探讨ddPCR检测病原菌载量值与感染指标水平的相关性。结果共纳入22例患者,采集52份静脉血标本进行检测,BC阳性17份(32.7%),检出病原体29株;ddPCR阳性41份(78.8%),检测出病原体73株。ddPCR耗时低于BC[(0.16±0.03)d VS(5.92±1.20)d,P<0.001]。在ddPCR检测范围内,以BC为金标准,ddPCR检测的灵敏度和特异度分别为80.0%、28.6%;联合检测前1周内非血标本微生物证据综合判定BSI,ddPCR检测的灵敏度和特异度分别提高至91.9%、76.9%。ddPCR耐药基因检测中,19份检出bla KPC,9份检出bla NDM/IMP,6份检出V an A/V an M,5份检出mec A。相关性分析显示病原菌载量值与C反应蛋白、降钙素原水平呈正相关(r分别为0.347、0.414,均P<0.05)。结论ddPCR作为一种辅助BC诊断BSI的检测方法具有灵敏度高、耗时低等优势,值得进一步探讨其在临床中的应用。展开更多
Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of ti...Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of tissue, the presence of disease, and the administration of therapeutic treatment. The aim of the present study was to identify optimal RGs in a set of blood samples collected at different time points (0, 24, 48, 72 h) from horses following administration of extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time quantitative polymerase chain reaction (qPCR). An SAS program developed on the algorithm of geNorm, SASqPCR, was used to determine stability of the expression and the number of optimal RGs. The results showed that the range of quantification cycle (Cq) values of the evaluated genes varied between 17 and 26 cycles, and that one optimal RG, ACTB, was sufficient for normalization of gene expression. Results of stability of expression demonstrated that ACTB was the optimal choice for all the samples studied. Notably, in samples collected at 72 h post ESWT, TBP showed a significant change in the expression level, and was not suitable for use as a RG. These results substantiate the importance of validating and selecting an appropriate RG.展开更多
文摘We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
文摘目的探讨微滴数字聚合酶链反应(ddPCR)在重症急性胰腺炎(SAP)合并疑似血流感染(BSI)病原学诊断中的价值。方法选取2022年7—9月某院重症医学科收治的SAP患者,在疑似BSI发作时同步采集静脉血进行ddPCR检测和血培养(BC)及药敏试验(AST),记录两种检测方法的耗时,比较ddPCR与BC的检测结果,计算ddPCR的病原学诊断效能,并探讨ddPCR检测病原菌载量值与感染指标水平的相关性。结果共纳入22例患者,采集52份静脉血标本进行检测,BC阳性17份(32.7%),检出病原体29株;ddPCR阳性41份(78.8%),检测出病原体73株。ddPCR耗时低于BC[(0.16±0.03)d VS(5.92±1.20)d,P<0.001]。在ddPCR检测范围内,以BC为金标准,ddPCR检测的灵敏度和特异度分别为80.0%、28.6%;联合检测前1周内非血标本微生物证据综合判定BSI,ddPCR检测的灵敏度和特异度分别提高至91.9%、76.9%。ddPCR耐药基因检测中,19份检出bla KPC,9份检出bla NDM/IMP,6份检出V an A/V an M,5份检出mec A。相关性分析显示病原菌载量值与C反应蛋白、降钙素原水平呈正相关(r分别为0.347、0.414,均P<0.05)。结论ddPCR作为一种辅助BC诊断BSI的检测方法具有灵敏度高、耗时低等优势,值得进一步探讨其在临床中的应用。
文摘Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of tissue, the presence of disease, and the administration of therapeutic treatment. The aim of the present study was to identify optimal RGs in a set of blood samples collected at different time points (0, 24, 48, 72 h) from horses following administration of extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time quantitative polymerase chain reaction (qPCR). An SAS program developed on the algorithm of geNorm, SASqPCR, was used to determine stability of the expression and the number of optimal RGs. The results showed that the range of quantification cycle (Cq) values of the evaluated genes varied between 17 and 26 cycles, and that one optimal RG, ACTB, was sufficient for normalization of gene expression. Results of stability of expression demonstrated that ACTB was the optimal choice for all the samples studied. Notably, in samples collected at 72 h post ESWT, TBP showed a significant change in the expression level, and was not suitable for use as a RG. These results substantiate the importance of validating and selecting an appropriate RG.
