中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
9期
843-850
,共8页
肝炎病毒,乙型%DNA,病毒%磁力学
肝炎病毒,乙型%DNA,病毒%磁力學
간염병독,을형%DNA,병독%자역학
Hepatitis B virus%DNA,viral%Magnetics
目的 建立一种基于磁珠法的快捷、简便、高纯度血液HBV DNA提取方法(以下简称磁珠法),为用于临床自动化定量检测的核酸诊断试剂研发提供技术支持.方法 用科华生物公司内部5份HBV强阳性血清标本,先通过磁珠的磁性、悬浮性、HBV DNA提取效果等对4种磁珠(A～D磁珠)进行筛选,并对磁珠用量(1 ～ 1.75μl/ml)、裂解液pH值(4 ～9)、洗涤液中是否加入乙醇和洗脱液带磁珠扩增等条件进行系统优化；并对建立的磁珠法线性范围、精密度、灵敏度、抗污染和抗干扰能力等技术性能进行评价.然后,分别用磁珠法与国内成熟的煮沸法定量检测100份HBV血清标本,用磁珠法与国际“金标准”Roche COBAS TaqMan Test(以下简称Roche法)定量检测50份HBV血清标本,并比较分析3种方法的相关性.结果 通过实验条件优选试验确定超顺磁A磁珠在物理性能和DNA提取效果都优于其他3种磁珠,磁珠最佳使用量为1.25 μl/ml,裂解液最佳pH值为7～8,洗涤液中可去除乙醇成分,并可直接带磁珠进行扩增反应.通过平行检测5份公司内部HBV强阳性血清标本以及梯度稀释的血清标本确定磁珠法的线性范围为4×101 ～4 ×108 IU/ml,煮沸法为4×102 ～4×108 IU/ml;磁珠法检测灵敏度为30 IU/ml,高于煮沸法(100 IU/ml)；在低浓度HBV血清标本检测中,磁珠法的精密度(CV为6.0％)优于煮沸法(CV为11.9％)；磁珠法检测的假阳性率为0,而煮沸法假阳性率为3.1％,磁珠法抗污染能力优于煮沸法；煮沸法在标本中肝素钠含量达到62.5 U/ml时,检出血清中HBV DNA含量明显下降,当肝素钠达1000 U/ml时,无法检出HBV DNA,而肝素钠对磁珠法几乎无影响.同时,磁珠法和煮沸法平行检测100份临床HBV血清标本,磁珠法的阳性率(86％)高于煮沸法(74％),且2种方法的相关性较好(R2=0.948,P＜0.05)；磁珠法和Roche法平行检测50份临床HBV血清标本,磁珠法检出阳性率[95％ (49/50)]与Roche法[100％( 50/50)]接近,2种方法间有显著相关性(R2=0.963,P＜0.05).结论 建立的磁珠法定量检测HBV DNA的得率高,具有较宽的检测范围、定量准确、操作简单,可作为实验室首选的核酸提取方法用于临床乙型肝炎诊断.
目的 建立一種基于磁珠法的快捷、簡便、高純度血液HBV DNA提取方法(以下簡稱磁珠法),為用于臨床自動化定量檢測的覈痠診斷試劑研髮提供技術支持.方法 用科華生物公司內部5份HBV彊暘性血清標本,先通過磁珠的磁性、懸浮性、HBV DNA提取效果等對4種磁珠(A～D磁珠)進行篩選,併對磁珠用量(1 ～ 1.75μl/ml)、裂解液pH值(4 ～9)、洗滌液中是否加入乙醇和洗脫液帶磁珠擴增等條件進行繫統優化；併對建立的磁珠法線性範圍、精密度、靈敏度、抗汙染和抗榦擾能力等技術性能進行評價.然後,分彆用磁珠法與國內成熟的煮沸法定量檢測100份HBV血清標本,用磁珠法與國際“金標準”Roche COBAS TaqMan Test(以下簡稱Roche法)定量檢測50份HBV血清標本,併比較分析3種方法的相關性.結果 通過實驗條件優選試驗確定超順磁A磁珠在物理性能和DNA提取效果都優于其他3種磁珠,磁珠最佳使用量為1.25 μl/ml,裂解液最佳pH值為7～8,洗滌液中可去除乙醇成分,併可直接帶磁珠進行擴增反應.通過平行檢測5份公司內部HBV彊暘性血清標本以及梯度稀釋的血清標本確定磁珠法的線性範圍為4×101 ～4 ×108 IU/ml,煮沸法為4×102 ～4×108 IU/ml;磁珠法檢測靈敏度為30 IU/ml,高于煮沸法(100 IU/ml)；在低濃度HBV血清標本檢測中,磁珠法的精密度(CV為6.0％)優于煮沸法(CV為11.9％)；磁珠法檢測的假暘性率為0,而煮沸法假暘性率為3.1％,磁珠法抗汙染能力優于煮沸法；煮沸法在標本中肝素鈉含量達到62.5 U/ml時,檢齣血清中HBV DNA含量明顯下降,噹肝素鈉達1000 U/ml時,無法檢齣HBV DNA,而肝素鈉對磁珠法幾乎無影響.同時,磁珠法和煮沸法平行檢測100份臨床HBV血清標本,磁珠法的暘性率(86％)高于煮沸法(74％),且2種方法的相關性較好(R2=0.948,P＜0.05)；磁珠法和Roche法平行檢測50份臨床HBV血清標本,磁珠法檢齣暘性率[95％ (49/50)]與Roche法[100％( 50/50)]接近,2種方法間有顯著相關性(R2=0.963,P＜0.05).結論 建立的磁珠法定量檢測HBV DNA的得率高,具有較寬的檢測範圍、定量準確、操作簡單,可作為實驗室首選的覈痠提取方法用于臨床乙型肝炎診斷.
목적 건립일충기우자주법적쾌첩、간편、고순도혈액HBV DNA제취방법(이하간칭자주법),위용우림상자동화정량검측적핵산진단시제연발제공기술지지.방법 용과화생물공사내부5빈HBV강양성혈청표본,선통과자주적자성、현부성、HBV DNA제취효과등대4충자주(A～D자주)진행사선,병대자주용량(1 ～ 1.75μl/ml)、렬해액pH치(4 ～9)、세조액중시부가입을순화세탈액대자주확증등조건진행계통우화；병대건립적자주법선성범위、정밀도、령민도、항오염화항간우능력등기술성능진행평개.연후,분별용자주법여국내성숙적자비법정량검측100빈HBV혈청표본,용자주법여국제“금표준”Roche COBAS TaqMan Test(이하간칭Roche법)정량검측50빈HBV혈청표본,병비교분석3충방법적상관성.결과 통과실험조건우선시험학정초순자A자주재물이성능화DNA제취효과도우우기타3충자주,자주최가사용량위1.25 μl/ml,렬해액최가pH치위7～8,세조액중가거제을순성분,병가직접대자주진행확증반응.통과평행검측5빈공사내부HBV강양성혈청표본이급제도희석적혈청표본학정자주법적선성범위위4×101 ～4 ×108 IU/ml,자비법위4×102 ～4×108 IU/ml;자주법검측령민도위30 IU/ml,고우자비법(100 IU/ml)；재저농도HBV혈청표본검측중,자주법적정밀도(CV위6.0％)우우자비법(CV위11.9％)；자주법검측적가양성솔위0,이자비법가양성솔위3.1％,자주법항오염능력우우자비법；자비법재표본중간소납함량체도62.5 U/ml시,검출혈청중HBV DNA함량명현하강,당간소납체1000 U/ml시,무법검출HBV DNA,이간소납대자주법궤호무영향.동시,자주법화자비법평행검측100빈림상HBV혈청표본,자주법적양성솔(86％)고우자비법(74％),차2충방법적상관성교호(R2=0.948,P＜0.05)；자주법화Roche법평행검측50빈림상HBV혈청표본,자주법검출양성솔[95％ (49/50)]여Roche법[100％( 50/50)]접근,2충방법간유현저상관성(R2=0.963,P＜0.05).결론 건립적자주법정량검측HBV DNA적득솔고,구유교관적검측범위、정량준학、조작간단,가작위실험실수선적핵산제취방법용우림상을형간염진단.
Objective To facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay,a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads.Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process,for the method development,screening and selecting the magnetic beads based on magnetism,suspension power and extraction effects of HBV DNA,the optimization of the selected key components of extraction reagents were performed,which including the quantity of magnetic beads( 1 - 1.75 μl/ml),the buffer pH of lysis (4 -9),washing conditions with or without alcohol,elution for proliferation with or without magnetic beads,and etc.The evaluation for the extraction method was carried out including linearity,precision,sensitivity,specificity,anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples,the magnetic beads method was also compared with the interntional "Gold standard" Roche COBAS Tagman test method ( Roche method) in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression.Results Optimization experiment result showed superparamagnetic beads A was the best among the candidates ( C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml,buffer pH 7 - 8 of lysis,ethanol was eliminated from washing buffer,proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4 × 101 -4 × 108 IU/ml and that of boiling method was 4 × 102 -4 × 108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0％ and boiling method was 11.9％.The false positive rate was 0 for beads method vs 3.1％ for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However,sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86％ vs boiling method 74％.The correlation between the two method was R2 =0.948,P ＜ 0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95％ (49/50) vs Roche method was 100％ (50/50).The correlation was R2 =0.963,P ＜ 0.05.Conclusions The established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction,broader testing range,accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.( Chin J Lab Med,2012,35:843 -850 )
