BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(...BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.展开更多
Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without n...Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.展开更多
The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we esta...The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.展开更多
基金Supported by the National Key Research and Development Program of China,No.2020YFC2002700the National Natural Science Foundation of China,No.81972010+1 种基金the CAMS Initiative for Innovative Medicine,No.2016-I2M-1-007the Science Developing Funds of Navy General Hospital,No.CXPY201810.
文摘BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.
基金supported by the National Key R&D Program of China(Nos.2021YFC2301900 and 2021YFC2301905)the National 13th Five-Year Grand Program on Key Infectious Disease Control(Nos.2018ZX10301-101 and 2018ZX10301101-001-001)+3 种基金the National Natural Science Foundation of China(Nos.82241072,82072271,and 82272319)the High-Level Public Health Specialized Talents Project of Beijing Municipal Health Commission(Nos.2022-2-018 and 2022-1-007)the Climbing the peak(Dengfeng)Talent Training Program of Beijing Hospitals Authority(No.DFL20191701)Beijing Key Laboratory for HIV/AIDS Research(No.BZ0089).
文摘Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.
基金supported by the National Natural Science Foundation of China (Nos. 31470271 and 81730110)Guangzhou Science and Technology Program key projects (No. 201803040006)
文摘The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
