Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV i...Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.展开更多
To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (...To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads展开更多
One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplifie...One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.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
目的建立SYBR Green Ⅰ荧光定量PCR技术测定HIV-1前病毒载量方法,并以其观察相关病毒进入抑制剂对HIV-1前病毒的作用。方法选择HIV-1的gag和env保守区域设计引物,建立SYBR Green I荧光定量PCR测定HIV-1前病毒载量的体系,并以其观察6μg/...目的建立SYBR Green Ⅰ荧光定量PCR技术测定HIV-1前病毒载量方法,并以其观察相关病毒进入抑制剂对HIV-1前病毒的作用。方法选择HIV-1的gag和env保守区域设计引物,建立SYBR Green I荧光定量PCR测定HIV-1前病毒载量的体系,并以其观察6μg/ml^6 ng/ml的重组病毒巨噬细胞炎症蛋白(rvMIP)、逆转录酶抑制剂(AZT)、膜融合抑制剂(T-20)、rvMIP+AZT、rvMIP+T-20对HIV-1前病毒载量的影响。结果所建立的荧光定量PCR体系的检测下限为101Copies,其标准曲线的相关系数为0.998 9,斜率为-4.925,截距为35.70;除6ng/ml的rvMIP、AZT、T-20外,三种药物处理后HIV-1前病毒载量均显著减小(P<0.05),并呈质量浓度依赖性;联合用药者前病毒载量显著低于单用rvMIP者(P<0.05),HIV-1前病毒载量rvMIP+T-20(rvMIP+AZT(rvMIP(T-20(AZT。结论本研究建立的SYBR Green Ⅰ荧光定量PCR检测方法具有灵敏、快速、准确及成本低廉等优点;与T-20或AZT联用可增强rvMIP对HIV-1前病毒的抑制作用。展开更多
文摘Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.
基金This project was supported by a grant from National Sci-ences Foundation of China( Serial No.3 93 70 0 3 8) and part ofa grant from Hubei Science and Technology Comm ittee( No.89JJ13 - 18)
文摘To find a technique of detecting and differentiating enteric adenoviruses (EAds) in clinical samples, a novel PCR approach was developed. EAds were able to be detected by use of a pair of subgroup F general primers (P1 and P2), and they were also be able to be differentiated from each other in the presence of another adenovirus type 40 (Ad40) specific primer (P3) in the same tube. Our results showed that there was one band for Ad41 and two bands for Ad40, respectively, on running-gel after PCR performance. PCR was performed on 40 specimens in parallel directly with dot-hybridization assay on the same diluted stool samples. 20 of 40 specimens were positive by hybridization (of them 12 were Ad41 and 8 were Ad40), whereas 26 were positive by PCR performance on the same samples with Ad41 18 and Ad40 8 positive as well. Our study indicated that this novel method could be used in clinical laboratory or in epidemic investigation for Eads
文摘One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.
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.
文摘目的建立SYBR Green Ⅰ荧光定量PCR技术测定HIV-1前病毒载量方法,并以其观察相关病毒进入抑制剂对HIV-1前病毒的作用。方法选择HIV-1的gag和env保守区域设计引物,建立SYBR Green I荧光定量PCR测定HIV-1前病毒载量的体系,并以其观察6μg/ml^6 ng/ml的重组病毒巨噬细胞炎症蛋白(rvMIP)、逆转录酶抑制剂(AZT)、膜融合抑制剂(T-20)、rvMIP+AZT、rvMIP+T-20对HIV-1前病毒载量的影响。结果所建立的荧光定量PCR体系的检测下限为101Copies,其标准曲线的相关系数为0.998 9,斜率为-4.925,截距为35.70;除6ng/ml的rvMIP、AZT、T-20外,三种药物处理后HIV-1前病毒载量均显著减小(P<0.05),并呈质量浓度依赖性;联合用药者前病毒载量显著低于单用rvMIP者(P<0.05),HIV-1前病毒载量rvMIP+T-20(rvMIP+AZT(rvMIP(T-20(AZT。结论本研究建立的SYBR Green Ⅰ荧光定量PCR检测方法具有灵敏、快速、准确及成本低廉等优点;与T-20或AZT联用可增强rvMIP对HIV-1前病毒的抑制作用。
