国际输血及血液学杂志
國際輸血及血液學雜誌
국제수혈급혈액학잡지
INTERNATIONAL JOURNAL OF BLOOD TRANSFUSION AND HEMATOLOGY
2014年
5期
435-439
,共5页
核酸扩增技术%聚合酶链反应%肝炎病毒,乙型%肝炎病毒,丙型%病毒,人类免疫缺陷
覈痠擴增技術%聚閤酶鏈反應%肝炎病毒,乙型%肝炎病毒,丙型%病毒,人類免疫缺陷
핵산확증기술%취합매련반응%간염병독,을형%간염병독,병형%병독,인류면역결함
Nucleic acid amplification techniques%Polymerase chain reaction%Hepatitis B virus%Hepatitis C virus%Human immunodeficiency virus-1
目的 初步探讨多重定量聚合酶链反应(PCR)同步检测乙型肝炎病毒(HBV) DNA,丙型肝炎病毒(HCV) RNA及人类免疫缺陷病毒(HIV)-1 RNA在血液筛查中的应用前景.方法 选择2012年8月至12月,于孝感市中心血站志愿献血的合格献血者血样中,经2次酶联免疫吸附法(ELISA)检测HBV表面抗原(HBsAg)、抗HCV及抗HIV-1,检测结果均呈阴性的4 800份血样为研究对象.采用全自动核酸混合提取仪对该4 800份血样进行核酸提取,然后利用多重定量PCR方法对血样中HBV、HCV及HIV-1进行同步扩增检测.采用中国药品和生物制品检定所提供的HBV DNA、HCV RNA及HIV-1 RNA标准参考品,检测多重定量PCR的灵敏度,并与单重定量PCR的灵敏度进行比较.在HBV、HCV及HIV-1 3者中任意1种病毒基因组浓度较高的条件下,对多重定量PCR检测另2种低浓度病毒基因组的能力进行评估.结果 增加多重定量PCR中c-MMLV逆转录酶和Hot Taq酶的用量,并适量加入单链结合蛋白(SSB),可使其扩增效率提升至单重定量PCR扩增水平.本组4 800份血样中,经多重定量PCR检测出3份HBV DNA阳性样品,ELISA漏检率为0.062 5％,未发现HCV RNA和HIV-1 RNA阳性样品;多重定量PCR检测HBV DNA、HCV RNA及HIV-1 RNA在95％置信区间的灵敏度浓度分别为115 IU/mL、376 IU /mL和232 IU /mL;单重定量PCR检测HBV DNA、HCV RNA和HIV-1 RNA在95％置信区间的灵敏度浓度分别为51 IU /mL、94 IU /mL和78 IU/mL.结论 本研究初步建立了对献血者血液同时进行HBV DNA、HCV RNA及HIV-1 RNA检测的多重定量PCR检测方法;该检测体系经过进一步优化后,有望应用于临床大规模血液病毒筛查.
目的 初步探討多重定量聚閤酶鏈反應(PCR)同步檢測乙型肝炎病毒(HBV) DNA,丙型肝炎病毒(HCV) RNA及人類免疫缺陷病毒(HIV)-1 RNA在血液篩查中的應用前景.方法 選擇2012年8月至12月,于孝感市中心血站誌願獻血的閤格獻血者血樣中,經2次酶聯免疫吸附法(ELISA)檢測HBV錶麵抗原(HBsAg)、抗HCV及抗HIV-1,檢測結果均呈陰性的4 800份血樣為研究對象.採用全自動覈痠混閤提取儀對該4 800份血樣進行覈痠提取,然後利用多重定量PCR方法對血樣中HBV、HCV及HIV-1進行同步擴增檢測.採用中國藥品和生物製品檢定所提供的HBV DNA、HCV RNA及HIV-1 RNA標準參攷品,檢測多重定量PCR的靈敏度,併與單重定量PCR的靈敏度進行比較.在HBV、HCV及HIV-1 3者中任意1種病毒基因組濃度較高的條件下,對多重定量PCR檢測另2種低濃度病毒基因組的能力進行評估.結果 增加多重定量PCR中c-MMLV逆轉錄酶和Hot Taq酶的用量,併適量加入單鏈結閤蛋白(SSB),可使其擴增效率提升至單重定量PCR擴增水平.本組4 800份血樣中,經多重定量PCR檢測齣3份HBV DNA暘性樣品,ELISA漏檢率為0.062 5％,未髮現HCV RNA和HIV-1 RNA暘性樣品;多重定量PCR檢測HBV DNA、HCV RNA及HIV-1 RNA在95％置信區間的靈敏度濃度分彆為115 IU/mL、376 IU /mL和232 IU /mL;單重定量PCR檢測HBV DNA、HCV RNA和HIV-1 RNA在95％置信區間的靈敏度濃度分彆為51 IU /mL、94 IU /mL和78 IU/mL.結論 本研究初步建立瞭對獻血者血液同時進行HBV DNA、HCV RNA及HIV-1 RNA檢測的多重定量PCR檢測方法;該檢測體繫經過進一步優化後,有望應用于臨床大規模血液病毒篩查.
목적 초보탐토다중정량취합매련반응(PCR)동보검측을형간염병독(HBV) DNA,병형간염병독(HCV) RNA급인류면역결함병독(HIV)-1 RNA재혈액사사중적응용전경.방법 선택2012년8월지12월,우효감시중심혈참지원헌혈적합격헌혈자혈양중,경2차매련면역흡부법(ELISA)검측HBV표면항원(HBsAg)、항HCV급항HIV-1,검측결과균정음성적4 800빈혈양위연구대상.채용전자동핵산혼합제취의대해4 800빈혈양진행핵산제취,연후이용다중정량PCR방법대혈양중HBV、HCV급HIV-1진행동보확증검측.채용중국약품화생물제품검정소제공적HBV DNA、HCV RNA급HIV-1 RNA표준삼고품,검측다중정량PCR적령민도,병여단중정량PCR적령민도진행비교.재HBV、HCV급HIV-1 3자중임의1충병독기인조농도교고적조건하,대다중정량PCR검측령2충저농도병독기인조적능력진행평고.결과 증가다중정량PCR중c-MMLV역전록매화Hot Taq매적용량,병괄량가입단련결합단백(SSB),가사기확증효솔제승지단중정량PCR확증수평.본조4 800빈혈양중,경다중정량PCR검측출3빈HBV DNA양성양품,ELISA루검솔위0.062 5％,미발현HCV RNA화HIV-1 RNA양성양품;다중정량PCR검측HBV DNA、HCV RNA급HIV-1 RNA재95％치신구간적령민도농도분별위115 IU/mL、376 IU /mL화232 IU /mL;단중정량PCR검측HBV DNA、HCV RNA화HIV-1 RNA재95％치신구간적령민도농도분별위51 IU /mL、94 IU /mL화78 IU/mL.결론 본연구초보건립료대헌혈자혈액동시진행HBV DNA、HCV RNA급HIV-1 RNA검측적다중정량PCR검측방법;해검측체계경과진일보우화후,유망응용우림상대규모혈액병독사사.
Objective To preliminarily explore the application prospects of multiple quantitative polymerase chain reaction (PCR) for synchronous detection of hepatitis B virus (HBV) DNA,hepatitis C virus (HCV) RNA and human immunodeficiency virus type 1 (HIV-1) RNA in blood screening.Methods From August to December 2012,a total of 4 800 qualified blood samples which were twice negative in enzyme linked immunosorbent assay (ELISA) screening for HBV surface antigen (HBsAg),HCV antibodies and HIV-1 antibody in Blood Center of Xiaogan,were collected into this study.Nucleic acids of all these samples were extracted automatically by Chemagic STAR sampling processor.A multiple quantitative PCR assay system for simultaneous detection,identification and quantification of HBV,HCV and HIV-1 was established and optimized.Using this system,the standard nucleic acids from National Institute for the Control of Pharmaceutical and Biological Products were detected to test sensitivity of multiple quantitative PCR.And its sensitivity was compared with single quantitative PCR.Then,the method for detecting a virus genome including rich nucleic acids of another kind of virus in this system was evaluated.Results The efficiency of multiple quantitative PCR was improved to the level of single quantitative PCR by increasing the addition of cMMLV reverse transcriptase and Hot-Taq enzyme and supplying with single-strand binding protein (SSB).Among 4 800 seronegative samples,3 samples were detected HBV DNA positive,and the positive rate was 0.062 5 ％.No HCV RNA positive and HIV-1 RNA positive were detected in this assay.In 95％ detection limits,sensitivity concentration of multiple quantitative PCR were 115 IU/mL,376 IU/mL and 232 IU/mL for HBV DNA,HCV RNA and HIV-1 RNA respectively,while those of the single quantitative PCR were 51 IU/mL,94 IU/mL and 78 IU/mL for HBV DNA,HCV RNA and HIV 1 RNA respectively.Conclusions A multiple quantitative PCR assay for simultaneous detection,identification and quantification of HBV,HCV and HIV-1 is developed.With further optimization,it may be expected to be applied to clinical massive blood screening.
