中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2013年
4期
333-338
,共6页
肝炎病毒,乙型%DNA,病毒%基因型%聚合酶链反应
肝炎病毒,乙型%DNA,病毒%基因型%聚閤酶鏈反應
간염병독,을형%DNA,병독%기인형%취합매련반응
Hepatitis B virus%DNA,viral%Genotype%Polymerase chain reaction
目的 研究建立同时进行HBV DNA定量与基因分型检测的双重分子信标实时PCR法(以下简称“定量与分型PCR法”),并探讨其应用价值.方法 构建B、C基因型重组质粒作为标准品,通过设计B、C基因型特异性引物和分子信标(B、C基因型分子信标5’端分别标记FAM和Hex荧光基团),建立在1个反应体系中同时定量与分型PCR法.然后,用10倍梯度稀释的B、C基因型标准品(103 ～ 1011 kIU/L)评价其检测线性范围和灵敏度；用丙型肝炎病毒感染者、单纯疱疹病毒感染者、人类乳头瘤病毒感染者、健康志愿者血清各5份评价其检测特异性；用高、中、低3份不同浓度的B、C基因型质粒标准品(108、106、104 kIU/L)进行同批次和不同批次各10次重复检测,分别计算批内及批间Ct值的变异系数(CV)评价其检测重复性.然后,以湖南圣湘生物科技有限公司HBV核酸定量测定试剂盒为HBV DNA定量参照方法,申友公司HBV基因分型试剂盒为HBV基因分型参照方法,同定量与分型PCR法平行检测132例HBV感染者血清标本,评价自建定量和分型PCR法的准确性.最后,将132例HBV感染者分为无症状携带组(21例)、慢性乙型肝炎组(77例)、肝硬化组(25例)、肝癌组(9例),分析评价基因型、疾病进展的不同阶段与DNA载量间的相关性.结果 用自建定量与分型PCR法检测B、C基因型的灵敏度均为103 kIU/L;线性范围均为103 ～ 1011 kIU/L;检测B基因型批内CV为1.51％ ～ 1.80％,批间CV为2.11％ ～3.03％,检测C基因型批内CV为1.79％～1.95％,批间CV为2.53％ ～2.91％；对其他病毒感染者和健康志愿者血清检测结果均为阴性.定量与分型PCR法检测132例HBV感染者的基因分型结果与HBV测序分型试剂盒总符合率为90.9％(120/132,Kappa=0.832,P＜0.05)；其DNA定量结果[5.07(3.89 ～6.33) lg kIU/L]与HBV核酸定量测定试剂盒定量结果[5.19(4.15 ～6.32) lg kIU/L]有很好的相关性(R2=0.8477,P＜0.05).此外,定量与分型PCR法检出B基因型69例、C基因型51例、B/C混合基因型12例,DNA载量分别为4.54(3.83 ～6.17)、5.53(4.02 ～6.55)、4.58 (3.68～4.98) lg kIU/L,C基因型感染者DNA水平高于B基因型和B/C混合基因型感染者(Z值分别为-2.195、-2.162,P均＜0.05)；无症状携带组、慢性乙型肝炎组、肝硬化组、肝癌组DNA载量分别为7.02(6.35 ～7.84)、4.94(4.16～6.25)、4.37(3.50 ～5.17)、3.45 (3.25 ～4.92) lg kIU/L,无症状携带组DNA水平显著高于其他3组(Z值分别为-4.244、-4.568、-3.489,P均＜0.001),慢乙型肝炎组DNA水平与肝硬化和肝癌组比较,差异均有统计学意义(Z值分别为-2.894、-2.413,P均＜0.05),但肝硬化组与肝癌组的差异无统计学意义(Z=-0.995,P=0.335).结论 建立的定量与分型PCR法准确、灵敏、特异,可同时进行HBV DNA的定量和基因分型检测.(中华检验医学杂志,2013,36:333-338
目的 研究建立同時進行HBV DNA定量與基因分型檢測的雙重分子信標實時PCR法(以下簡稱“定量與分型PCR法”),併探討其應用價值.方法 構建B、C基因型重組質粒作為標準品,通過設計B、C基因型特異性引物和分子信標(B、C基因型分子信標5’耑分彆標記FAM和Hex熒光基糰),建立在1箇反應體繫中同時定量與分型PCR法.然後,用10倍梯度稀釋的B、C基因型標準品(103 ～ 1011 kIU/L)評價其檢測線性範圍和靈敏度；用丙型肝炎病毒感染者、單純皰疹病毒感染者、人類乳頭瘤病毒感染者、健康誌願者血清各5份評價其檢測特異性；用高、中、低3份不同濃度的B、C基因型質粒標準品(108、106、104 kIU/L)進行同批次和不同批次各10次重複檢測,分彆計算批內及批間Ct值的變異繫數(CV)評價其檢測重複性.然後,以湖南聖湘生物科技有限公司HBV覈痠定量測定試劑盒為HBV DNA定量參照方法,申友公司HBV基因分型試劑盒為HBV基因分型參照方法,同定量與分型PCR法平行檢測132例HBV感染者血清標本,評價自建定量和分型PCR法的準確性.最後,將132例HBV感染者分為無癥狀攜帶組(21例)、慢性乙型肝炎組(77例)、肝硬化組(25例)、肝癌組(9例),分析評價基因型、疾病進展的不同階段與DNA載量間的相關性.結果 用自建定量與分型PCR法檢測B、C基因型的靈敏度均為103 kIU/L;線性範圍均為103 ～ 1011 kIU/L;檢測B基因型批內CV為1.51％ ～ 1.80％,批間CV為2.11％ ～3.03％,檢測C基因型批內CV為1.79％～1.95％,批間CV為2.53％ ～2.91％；對其他病毒感染者和健康誌願者血清檢測結果均為陰性.定量與分型PCR法檢測132例HBV感染者的基因分型結果與HBV測序分型試劑盒總符閤率為90.9％(120/132,Kappa=0.832,P＜0.05)；其DNA定量結果[5.07(3.89 ～6.33) lg kIU/L]與HBV覈痠定量測定試劑盒定量結果[5.19(4.15 ～6.32) lg kIU/L]有很好的相關性(R2=0.8477,P＜0.05).此外,定量與分型PCR法檢齣B基因型69例、C基因型51例、B/C混閤基因型12例,DNA載量分彆為4.54(3.83 ～6.17)、5.53(4.02 ～6.55)、4.58 (3.68～4.98) lg kIU/L,C基因型感染者DNA水平高于B基因型和B/C混閤基因型感染者(Z值分彆為-2.195、-2.162,P均＜0.05)；無癥狀攜帶組、慢性乙型肝炎組、肝硬化組、肝癌組DNA載量分彆為7.02(6.35 ～7.84)、4.94(4.16～6.25)、4.37(3.50 ～5.17)、3.45 (3.25 ～4.92) lg kIU/L,無癥狀攜帶組DNA水平顯著高于其他3組(Z值分彆為-4.244、-4.568、-3.489,P均＜0.001),慢乙型肝炎組DNA水平與肝硬化和肝癌組比較,差異均有統計學意義(Z值分彆為-2.894、-2.413,P均＜0.05),但肝硬化組與肝癌組的差異無統計學意義(Z=-0.995,P=0.335).結論 建立的定量與分型PCR法準確、靈敏、特異,可同時進行HBV DNA的定量和基因分型檢測.(中華檢驗醫學雜誌,2013,36:333-338
목적 연구건립동시진행HBV DNA정량여기인분형검측적쌍중분자신표실시PCR법(이하간칭“정량여분형PCR법”),병탐토기응용개치.방법 구건B、C기인형중조질립작위표준품,통과설계B、C기인형특이성인물화분자신표(B、C기인형분자신표5’단분별표기FAM화Hex형광기단),건립재1개반응체계중동시정량여분형PCR법.연후,용10배제도희석적B、C기인형표준품(103 ～ 1011 kIU/L)평개기검측선성범위화령민도；용병형간염병독감염자、단순포진병독감염자、인류유두류병독감염자、건강지원자혈청각5빈평개기검측특이성；용고、중、저3빈불동농도적B、C기인형질립표준품(108、106、104 kIU/L)진행동비차화불동비차각10차중복검측,분별계산비내급비간Ct치적변이계수(CV)평개기검측중복성.연후,이호남골상생물과기유한공사HBV핵산정량측정시제합위HBV DNA정량삼조방법,신우공사HBV기인분형시제합위HBV기인분형삼조방법,동정량여분형PCR법평행검측132례HBV감염자혈청표본,평개자건정량화분형PCR법적준학성.최후,장132례HBV감염자분위무증상휴대조(21례)、만성을형간염조(77례)、간경화조(25례)、간암조(9례),분석평개기인형、질병진전적불동계단여DNA재량간적상관성.결과 용자건정량여분형PCR법검측B、C기인형적령민도균위103 kIU/L;선성범위균위103 ～ 1011 kIU/L;검측B기인형비내CV위1.51％ ～ 1.80％,비간CV위2.11％ ～3.03％,검측C기인형비내CV위1.79％～1.95％,비간CV위2.53％ ～2.91％；대기타병독감염자화건강지원자혈청검측결과균위음성.정량여분형PCR법검측132례HBV감염자적기인분형결과여HBV측서분형시제합총부합솔위90.9％(120/132,Kappa=0.832,P＜0.05)；기DNA정량결과[5.07(3.89 ～6.33) lg kIU/L]여HBV핵산정량측정시제합정량결과[5.19(4.15 ～6.32) lg kIU/L]유흔호적상관성(R2=0.8477,P＜0.05).차외,정량여분형PCR법검출B기인형69례、C기인형51례、B/C혼합기인형12례,DNA재량분별위4.54(3.83 ～6.17)、5.53(4.02 ～6.55)、4.58 (3.68～4.98) lg kIU/L,C기인형감염자DNA수평고우B기인형화B/C혼합기인형감염자(Z치분별위-2.195、-2.162,P균＜0.05)；무증상휴대조、만성을형간염조、간경화조、간암조DNA재량분별위7.02(6.35 ～7.84)、4.94(4.16～6.25)、4.37(3.50 ～5.17)、3.45 (3.25 ～4.92) lg kIU/L,무증상휴대조DNA수평현저고우기타3조(Z치분별위-4.244、-4.568、-3.489,P균＜0.001),만을형간염조DNA수평여간경화화간암조비교,차이균유통계학의의(Z치분별위-2.894、-2.413,P균＜0.05),단간경화조여간암조적차이무통계학의의(Z=-0.995,P=0.335).결론 건립적정량여분형PCR법준학、령민、특이,가동시진행HBV DNA적정량화기인분형검측.(중화검험의학잡지,2013,36:333-338
Objective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR.Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype.The molecular beacons of genotype B and C were labeled with FAM and Hex respectively.In this way,a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed.Firstly,10-fold gradient dilution of genotype B and C standard plasmids (103-1011 kIU/L) were utilized to evaluate the linear ranges and sensitivity of this approach.The clinical specificity was tested with twenty different serum specimens (5 cases with hepatitis C virus,5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers) ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation (CV) of cycle threshold (Ct) value through 10 repeated detections within a batch and between batches of the B,C standard plasmids (108,106 and 104 kIU/L).Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references.Finally,these HBV-positive patients were divided into 4 groups:asymptomatic carrier (n =21),chronic hepatitis (n =77),liver cirrhosis (n =25) and hepatocellular carcinoma (n =9) to investigate the relationship of genotypes,stages of disease progression and HBV DNA load.Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103 kIU/L and the linear range was 103-1011 kIU/L.The intra-assay CV of B genotyping was 1.51％ to 1.80％ and the interassay CV was 2.11％ to 3.03％,while the intra-assay CV of C genotyping was 1.79％ to 1.95％ and the inter-assay CV was 2.53％ to 2.91％.The results of non HBV infected cases and healthy volunteers showed negative.In the test of 132 HBV infected patients,the general coincident rate of genotyping results comparing our assay and HBV DNA genotyping kit was 90.9％ (120/132,Kappa =0.832,P ＜ 0.05).The HBV DNA quatitive results between the assay[5.07 (3.89-6.33)] and HBV DNA quatitive kit [5.19 (4.15-6.32) lg kIU/L] were well correlative (R2 =0.8477,P ＜ 0.05).69 genotype B cases,51 genotype C cases and 12 B/C mixed-genotype cases were detected by dual molecular beacon real-time PCR method and their HBV DNA load were 4.54 (3.83-6.17),5.53 (4.02-6.55),4.58 (3.68-4.98) lg kIU/L respectively.Where the patients with genotype C had higher DNA load than the patients with other two genotypes (Z =-2.195and-2.162,P ＜ 0.05).The HBV DNA load of asymptomatic group,chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group were 7.02 (6.35-7.84),4.94 (4.16-6.25),4.37(3.50-5.17) and 3.45 (3.25-4.92) lg kIU/L,respectively.Among them,the asymptomatic group was significantly higher than those of other three groups (Z =-4.244,-4.568 and-3.489,P ＜0.001) and DNA load comparing with the chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group also showed statistically different (Z =-2.894 and-2.413,P ＜ 0.05).However,compared with the liver cirrhosis group and hepatocellular carcinoma group there was no significant difference (Z =-0.995,P =0.335).Conclusion A dual molecular beacon real-time PCR assay which can simultaneously quantifying and genotyping HBV DNA with highly accuracy,sensitive and specificity is successfully developed.(Chin J Lab Med,2013,36:333-338)
