Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essentia...Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.展开更多
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
DPO is one of the new specific primers for virus detection.In this study,five sets of dual priming oligonucleotide(DPO) primers for Potato virus X(PVX),Potato virus Y(PVY),Potato virus V(PVV),Tobacco ring spot virus(T...DPO is one of the new specific primers for virus detection.In this study,five sets of dual priming oligonucleotide(DPO) primers for Potato virus X(PVX),Potato virus Y(PVY),Potato virus V(PVV),Tobacco ring spot virus(TRSV) and Cucumber mosaic virus(CMV) were designed and applied to the detection of the main potato viruses with multiplex reverse transcription polymerase chain reaction(m-RT-PCR),including two quarantine viruses in China(PVV and TRSV).The results showed that DPO-m-RT-PCR exhi-bited high PCR specificity and sensitivity even under less than optimal PCR condition compared with conventional m-RT-PCR.DPO-m-RT-PCR could be applied in the healthy evaluation of seed potato and its exit-entry quarantine.展开更多
A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of three sweet potato potyviruses: Sweet potato feathery mottle vir...A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of three sweet potato potyviruses: Sweet potato feathery mottle virus(SPFMV),Sweet potato latent virus (SPLV) and Sweet potato virus G (SPVG). Three compatible sets of primers specific for each virus were designed in conserved regions of the coat protein (CP) gene for use in multiplex RT-PCR assay, and producing three distinct fragments 300, 420, and 600 bp, indicating the presence of SPFMV, SPLV and SPVG respectively. The individual RT-PCR assays and the multiplex assay were optimized for highest sensitivity and specificity. This study fulfilled the need for rapid and specific sweet potato potyvirus diagnostic tool and that also had the potential for investigating the epidemiology of sweet viral diseases.展开更多
文摘Introduction: Arbovirus diseases such as dengue and chikungunya threaten public health worldwide. Early and rapid diagnosis and surveillance of dengue virus (DENV) and chikungunya virus (CHIKV) infections are essential to the control of these diseases. In this study, we evaluate the diagnostic performance of our new in-house multiplex RT-qPCR method for detecting DENV serotypes and CHIKV in an external laboratory. Methodology: The evaluation study was conducted on 200 clinical samples of suspected patients for arbovirus disease infection, collected in Centre de Recherche Biomoléculaire Pietro Annigoni (CERBA), Ouagadougou, Burkina Faso. Our new multiplex RT-qPCR was compared to the commercial kit, the Zika, Dengue, and Chikungunya (ZDC) Real-Time PCR Assays kit (Bio-Rad, California, USA). Results and Conclusions: Among 200 samples, 21.5% (43/200) were DENV-positive by multiplex RT-qPCR, and 21.5% (43/200) were also DENV-positive by reference real-time RT-PCR. 157 (78.5%) samples tested negative for DENV by both tests (new mRT-qPCR and reference test). The sensitivity and specificity of mRT-qPCR were 100%. The DENV serotypes detected were DENV-1 60.5% (26/43) and DENV-3 39.5% (17/43). CHIKV was not detected in this study. Our new mRT-qPCR is sensitive, cost-effective, simple, and can be used in developing country laboratories.
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DPO is one of the new specific primers for virus detection.In this study,five sets of dual priming oligonucleotide(DPO) primers for Potato virus X(PVX),Potato virus Y(PVY),Potato virus V(PVV),Tobacco ring spot virus(TRSV) and Cucumber mosaic virus(CMV) were designed and applied to the detection of the main potato viruses with multiplex reverse transcription polymerase chain reaction(m-RT-PCR),including two quarantine viruses in China(PVV and TRSV).The results showed that DPO-m-RT-PCR exhi-bited high PCR specificity and sensitivity even under less than optimal PCR condition compared with conventional m-RT-PCR.DPO-m-RT-PCR could be applied in the healthy evaluation of seed potato and its exit-entry quarantine.
文摘A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) method was developed for the simultaneous detection and discrimination of three sweet potato potyviruses: Sweet potato feathery mottle virus(SPFMV),Sweet potato latent virus (SPLV) and Sweet potato virus G (SPVG). Three compatible sets of primers specific for each virus were designed in conserved regions of the coat protein (CP) gene for use in multiplex RT-PCR assay, and producing three distinct fragments 300, 420, and 600 bp, indicating the presence of SPFMV, SPLV and SPVG respectively. The individual RT-PCR assays and the multiplex assay were optimized for highest sensitivity and specificity. This study fulfilled the need for rapid and specific sweet potato potyvirus diagnostic tool and that also had the potential for investigating the epidemiology of sweet viral diseases.
