亚甲蓝光化学法降解丙型肝炎病毒核酸的研究

目的研究亚甲蓝光化学法（methylene blue photochemistry,MB-P）降解病毒核酸的作用机制,为该方法在临床血液制品病毒灭活中的应用提供理论依据。方法以丙型肝炎病毒（HCV）为研究对象,选择HCV基因组中相对保守的5′-NCR为模板,应用荧光定量PCR技术观察不同处理时间HCV RNA降解情况。同时,对体外转录的同区段的HCV RNA在无蛋白成分的代血浆（羟乙基淀粉20-氯化钠注射液）及PBS缓冲液中的降解模式进行分析。结果MB-P处理条件下,HCV RNA含量呈对数形式下降,该方法对体外转录RNA的灭活效率低于对血浆中病毒颗粒的灭活效率。结论亚甲蓝能作用于病毒核酸,在光照条件下使RNA迅速降解,从而达到病毒灭活的效果。     

丙型肝炎病毒样颗粒作为亚甲蓝光化学法灭活HCV效果评价物的可行性研究

目的探讨丙型肝炎病毒样颗粒(HCVLPs)在亚甲蓝光化学灭活处理过程中的变化趋势及其作为亚甲蓝光化学法灭活HCV效果评价物的可行性。方法以HCV阳性血浆作为平行对照组(HCV RNA载量约6.53 logcopies/m L)(n=6),将HCVLPs用代血浆调整成合适浓度(HCV RNA定量约为6.74 logcopies/m L)的悬液作为实验组(n=5),将2组分装至PVC血袋中,MB+L 1μmol/L进行病毒灭活处理,分别于光照处理0、5、10、20、30 min时取样,用荧光定量PCR技术检测病毒核酸载量;同时以细胞病变法测定HCV模型病毒sindbis病毒的残余滴度,验证MB+L灭活HCV的效果。结果模型病毒sindbis的病毒滴度降至检测限以下(log TCID50/m L≤0.5);MB+L处理过程中,随着光照处理时间的增加,HCV及HCVLPs的核酸载量明显下降,分别从6.53与6.74下降至4.51和2.89log copies/m L(P0.05),且2组在不同取样点的HCV RNA载量之间具有相关性(R20.98,Significance F0.01)。结论 HCVLPs能够反映MB+L灭活处理中HCV RNA动态变化,或有望成为安全而有效的HCV灭活评价物来监控HCV灭活效果。     

亚甲蓝光化学法对红细胞膜的损伤

目的　为进一步完善和发展亚甲蓝光化学法 ( MB-P)在红细胞制品病毒灭活中的应用提供理论依据。方法　应用 MB-P法 (光照时间 60 min,MB浓度 5μmol/L,光强度 3 .8× 1 0 4 lx)处理人红细胞悬液后 ,用不同方法测定红细胞溶血度、膜脆性、膜乙酰胆碱酯酶 ( Ach E)活性、膜蛋白浓度、膜脂质流动性、膜脂质过氧化产物含量 ( MDA)、2 ,3 -DPG和 ATP含量。结果　 MB-P法对人红细胞有一定的损伤 ,损伤效应与 MB的浓度和光照时间有关。结论　 MB-P引起红细胞膜氧化损伤的分子机制主要是 1 O2 对膜脂的过氧化作用     

氯和二氧化氯灭活甲型肝炎病毒机理的研究

为探讨氯和二氧化氯灭活HAV机理及用PCR评价消毒效果的可行性,采用细胞培养、ELISA及大片段逐步步移RT-PCR方法对消毒前后HAV感染性、HAAg抗原性及HAV核酸全序列进行检测。结果,以含有效氯10 mg/L消毒液作用30 min,或含二氧化氯7.5 mg/L的消毒液作用10 min,对HAV感染性的灭活率均为100％,均可破坏HAV核酸5’非编码区;但检测两者HAAg抗原性P/N值分别为3.21(阳性)与2.02(阴性)。结果表明,HAV感染性的灭活与HAV核酸5’非编码区破坏是一致的,可用PCR技术检测HAV核酸5’非编码区以判定HAV灭活与否。     

经白细胞过滤和病毒灭活新鲜冰冻血浆制备冷沉淀的可行性探讨

目的探讨经白细胞过滤和亚甲蓝光化学法(methylene blue photochemistry,MB-P)病毒灭活后新鲜血浆制备冷沉淀凝血因子的有效成分Ⅷ因子和纤维蛋白原(Fib)含量的变化,为临床应用病毒灭活冷沉淀凝血因子进行治疗提供使用依据。方法对93袋全血(400ml)经白细胞过滤后分离制备的新鲜血浆进行MB-P病毒灭活制成病毒灭活冷沉淀,测定因子Ⅷ和纤维蛋白原的含量,同时与标准进行比较。结果 93袋经MB-P病毒灭活后新鲜冰冻血浆(FFP)制备的冷沉淀凝血因子中凝血因子(Ⅷ)平均值为71.28IU/袋,标准差18.60,90%区间分布范围为47.48~95.09IU/袋;纤维蛋白原(Fib)平均值为121.38IU/袋,标准差22.74,90%区间分布范围为92.28~150.49/袋,FⅧ和Fib含量未达到冷沉淀凝血因子的标准,但对FⅧ和Fib的影响符合相关报道。结论加强MB-P及制备过程控制,减少FⅧ和Fib损失,提高留存率,经MB-P病毒灭活后新鲜血浆制备冷沉淀凝血因子方法还是可行的,因MB-P病毒灭活新鲜血浆制备的冷沉淀凝血因子没有标准,暂不能提供临床。     

亚甲蓝／光照法病毒灭活血浆的制备及应用评价

目的探讨亚甲蓝／光照法病毒灭活血浆的制备及其临床应用评价。方法随机抽取200份病毒灭活血浆,检测灭活前后的血浆容量、总蛋白、凝血因子及亚甲蓝的残余量;选取15756人份HBsAg、抗-HCV、抗-HIV酶联免疫吸附法检测阴性者的血浆,在病毒灭活前后分别进行HBV、HCV、HIV的核酸检测;临床随机抽取200例输注病毒灭活血浆的患者,观察病毒灭活血浆输注后是否出现输血不良反应。结果经亚甲蓝／光照法进行灭活处理的血浆,血浆容量达到（227．34±5．21）g,回收率达到（96．67±2．34）％;血浆总蛋白、血浆凝血因子（Ⅷ因子和纤维蛋白原）回收率分别达到（88．69±3．32）％,（86．84±2．16）％和（84．62±1．86）％,与处理前相比,差异无统计学意义（P〉0．05）;血浆经病毒灭活后亚甲蓝的残余量为（1．17±0．05）μmol／L,而过滤后残余量减少至（0．16±0．03）μmol／L,去除率达到86．32％;对15756人份血浆进行HBV、HCV、HIV核酸检测,发现HBV阳性23例,HIV阳性1例,阳性率为1．52‰,进行病毒灭活后,HBV、HCV、HIV核酸检测均为阴性。病毒灭活血浆输注人体后无不良反应发生。结论采用亚甲蓝／光照法进行病毒灭活的血浆,临床使用较安全;病毒灭活血浆能够有效降低经输血传播疾病的危险性,且不良反应较小。     

运用FQ-PCR技术监测亚甲蓝光化学法对血浆丙型肝炎病毒的灭活效果

目的通过荧光定量PCR(FQ-PCR)技术对血浆病毒灭活前后丙型肝炎病毒RNA (HCV-RNA)浓度的测定,判断亚甲蓝(MB)光化学法灭活血浆丙型肝炎病毒的效果,为临床判别病毒灭活效果提供直接和客观依据.方法4份经证实为HCV-RNA阳性的血浆分别加入MB,终浓度为1μmol/L,经可见光(大于30000Lux)照射不同时间(0、5、10、15和30min)后,分别用FQ-PCR测定这些血浆的HCV-RNA浓度,并与未用MB处理的血浆作比较,判断随照射时间不同,HCV被灭活的效果.结果4份HCV-RNA阳性病人的血浆未经处理时其病毒核酸浓度分别为8.0×105拷贝/ml、1.7×106拷贝/ml、1.6×106拷贝/ml和3.1×105拷贝/ml,加入MB未经照射时HCV-RNA浓度即明显下降,可见光照射5min后HCV-RNA浓度分别下降为未经任何处理时浓度的18.75%、23.36%、4.66%和1.27%,随照射时间延长,病毒核酸浓度逐渐下降,照射30min后,HCV-RNA浓度均下降为零.结论用MB加光照30min处理,可达到将血浆丙肝病毒灭活的效果;FQ-PCR可定量检测MB光化学法灭活丙肝病毒的过程.     

热力与亚甲蓝光化学法联合应用灭活辛德毕斯病毒效果的研究

目的研究热力与亚甲蓝光化学法联合应用灭活辛德毕斯病毒的效果。方法采用细胞培养法和基因检测技术,对热力与亚甲蓝光化学法联合应用灭活辛德毕斯病毒的效果进行了评价。结果经56℃水浴中热力预灭活处理1.5 h,辛德毕斯病毒滴度≤0.5 TCID50/ml,灭活对数值达到6.33 TCID50/ml;未经预灭活的辛德毕斯病毒在1μmol/L亚甲蓝+光照(MB+L)处理中随光照时间的延长,病毒残余滴度逐渐下降至检测限(TCID50/ml=0.5)。经热力灭活预处理的辛德毕斯病毒与未经预处理的病毒核酸载量之间具有相关性(R20.98,显著性F0.01)。结论热力灭活预处理的辛德毕斯病毒能够反映活病毒的亚甲蓝病毒灭活效果,有望成为安全而有效的病毒灭活效果评价制品。     

血浆丙型肝炎病毒核酸稳定性的研究

目的：建立血浆丙型病毒性肝炎病毒(HCV)核酸的稳定保存方法，提高荧光定量聚合酶链反应(FQ-PCR)检测的准确性。方法：HCV混合血浆以TRIZOL提取病毒核酸，分别保存于70％乙醇溶液及溶解于焦碳酸二乙醇(DEPC)水中。在4℃放置不同时间后，以FQ-PCR检测HCV RNA拷贝数。HCV RNA冻干质控品溶解于DEPC水并提取核酸后同时测定作为对照。另外，对保存的HCV RNA稀释后作线性分析。结果：未提取核酸的病毒血浆及70％乙醇溶液中的HCV RNA在4℃保存8d后，病毒核酸拷贝数无明显改变。HCV RNA DEPC水溶液在4℃放置30min后即发现拷贝数降低,5h后下降为零。质控品测定值无明显降低。在70％乙醇溶液中保存的HCV RNA稀释后测定值具有线性(r=0．9991)。结论：病毒在血浆及70％乙醇溶液中保存，其核酸可以在4℃稳定保存至少8d并具有良好的线性。病毒核酸DEPC水溶液在4℃易发生降解。     

病毒灭活血浆亚甲蓝及凝血因子的质量控制

亚甲蓝光化学法（MB-P）可消毒处理单袋新鲜冰冻人血浆,对经血液传播的主要病毒HIV、HBV和HCV等都具灭活作用,能显著提高临床输血的安全性,已在我国推广应用,但对于该法制备的病毒灭活血浆还缺乏系统规范的操作方法和质量控制标准,     

丙型肝炎病毒总抗原检测用于病程监测的研究

目的探讨丙型肝炎病毒(HCV)总抗原检测方法在丙肝病程监测方面的临床意义。方法对来本院就诊的40位丙肝患者于治疗前、治疗1个月时、治疗3个月时、治疗6个月(停药)时,停药6个月后等不同时期进行采血,收集血清或血浆标本,用抗-HCV检测试剂盒(酶联免疫法)、HCV核酸(RNA)扩增(PCR)荧光定量检测试剂盒、HCV总抗原检测试剂盒(酶联免疫法)进行检测。结果从患者确认感染丙肝到治疗结束抗-HCV检测均呈阳性,而HCV-RNA检测和HCV总抗原检测会随着病程的变化而变化。本次共检测了189例标本(40位患者不同时期标本总例数),其中HCV-RNA阳性51例,该51例阳性标本中,HCV总抗原检测阳性44例,阳性检出率为86.27%;138例HCV-RNA阴性标本,有3例HCV总抗原检测为阳性(2.2%)。2种方法比较,差异无统计学意义(χ2=1.6,P>0.05)。HCV总抗原检测其OD值会随着病程的变化而相应改变,可以较好地反应丙肝患者的病程状况。结论 HCV总抗原检测方法在丙肝病程监测方面具有很好的临床意义,适合在缺少荧光定量PCR检测能力的中小医院使用,可在一定程度上替代HCV-RNA检测,对抗-HCV阳性患者作进一步的验证检测或补充,更好地应用于丙肝患者的病程监测。     

Analysis of the 5' end structure of HCV subgenomic RNA replicated in a Huh7 cell line

OBJECTIVE: Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5' end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate. METHODS: HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5' end. RESULTS: Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase. CONCLUSION: It is suggested that the 5' end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5' end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5' end. This result indicates that the 5' end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.     

亚甲蓝光化学法灭活血制品中巨细胞病毒的研究

目的探讨亚甲蓝光化学法(MBP)灭活血制品中巨细胞病毒(HCMV)的效果。方法将HCMVAD16910%分别加入血浆、红细胞悬液中并进行MBP病毒灭活处理(MB浓度5μmol/L,光照时间1h,光照强度38000lux),分别加至人胚肺成纤维细胞(HELF)单层中培养,与对照细胞比较观察细胞病变情况(CPE)。结果经MBP病毒灭活处理后,含HCMV血浆及红细胞的组织培养半数感染量(TCID50)下降4～6。结论MBP能灭活血液中的HCMV。     

Direct detection of circulating hepatitis C virus RNA using probes from the 5'' untranslated region.

Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards.     

丙肝病毒核心总抗原定量检测的临床应用

目的 探讨定量检测丙型肝炎病毒核心总抗原（total HCV-cAg,tHCV-cAg）在丙型肝炎诊断中的作用及与病毒载量的相关性.方法 随机选取广州军区武汉总医院2010年10月-2013年3月丙肝患者血清标本66例,分别采用化学发光法检测血清tHCV cAg,酶联免疫法（ELISA）检测血清丙型肝炎游离核心抗原（free HCV-cAg,fHCV-cAg）及丙型肝炎抗体（hepatitis C virus antibody,HCV-Ab）,实时荧光定量PCR方法检测血清HCV-RNA,全自动生化分析仪检测血清谷氨酸氨基转移酶（ALT）.比较HCV-RNA,tHCV-cAg及fHCV-cAg阳性率差异,同时分析tHCV-cAg与HCV-RNA及ALT相关性,探讨tHCV-cAg联合检测HCV-Ab与HCV-RNA检测符合率.结果 66例丙型肝炎患者血清中,tHCV-eAg阳性率与HCV-RNA阳性率差异无统计学意义（χ^2=0.165,P＞0.05）;tHCV-cAg量与HCV-RNA呈正相关：logHCV Ag=0.81 log HCV RNA-1.31（γ=0.83,P＜0.05）.tHCV-cAg阳性率与fHCV cAg阳性率差异有统计学意义（χ^2 =12.53,P＜0.05）.HCV-cAg阳性组ALT值异常率与HCV-eAg阴性组ALT值异常率差异有统计学意义（P＜0.05,χ^2=17.47）.tHCV-cAg联合检测HCV-Ab符合率与HCV-RNA检测达100.00％.结论 化学发光法定量检测血清tHCV-cAg阳性率显著高于fHCV-cAg,与HCV RNA阳性符合率达96.08％ （49/51）,是丙型肝炎早期诊断的特异性指标.tHCV-cAg和HCV-RNA呈正相关性,与肝功能损害相关,是反映病毒的复制指标.tHCV-cAg联合检测HCV-Ab可减低丙型肝炎的漏诊率.     

原料血浆混样HCV/HIV-1核酸检测的方法学研究

目的　建立原料血浆混浆核酸检测方法。方法　以国产HCV和HIV核酸扩增 (PCR)荧光定量检测试剂进行WHO标准品的灵敏度、重现性和精密度实验 ,并对 19196份国内原料血浆进行HCVRNA和HIV 1RNA核酸扩增分析。结果　扩增系统能够确保高拷贝数 (2 0 0IU/ml)标准品核酸的检出率 ,对于 <10 0IU/ml的低拷贝数标准品核酸检出率逐渐降低 ;受检原料血浆样本没有检出HCVRNA和HIV 1RNA阳性。结论　核酸扩增方法适用于原料血浆病毒筛查。     

Effect of heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) apheresis on hepatitis C plasma virus load.

Association of the hepatitis C virus (HCV) with apolipoprotein B containing lipoproteins has been suggested, and this led to the concept that the low-density lipoprotein (LDL) receptor may also serve as a candidate receptor for HCV uptake into the liver. We have investigated whether heparin-induced extracorporeal LDL precipitation (HELP) LDL apheresis treatment reduces HCV plasma load in 6 patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy. HELP apheresis treatment caused an HCV-RNA decrease of 77.3% in mean. This decline was not correlated with LDL-cholesterol reduction. HCV-RNA was retained on the HELP filter as shown for 1 patient. The effect of RNA lowering was only transient due to the high turnover of HCV. However, HELP apheresis may open a window of opportunity for an immune-modulating and antiviral therapy in the interval between two apheresis procedures in patients with high virus load.     

三种不同检测方法对婴幼儿丙型肝炎病毒感染的诊断价值

目的探讨不同检测方法对婴幼儿丙型肝炎病毒感染诊断结果的影响。方法对66例ELISA法检测抗HCV阳性孕妇所生婴幼儿血清,分别用ELISA法检测抗HCV和HCV-cAg,用荧光定量PCR法检测HCV-RNA,并对结果对比分析。结果 66例婴幼儿血清中,61例抗HCV阳性,阳性率92.4%;9例HCV-cAg阳性,阳性率13.6%;11例HCV-RNA阳性,阳性率16.6%。结论抗HCV检测阳性率过高,且有可能漏诊。建议采用HCV-RNA检测和HCV-cAg综合诊断婴幼儿HCV感染。     

荧光定量PCR与RT-PCR技术检测HCV-RNA的比较观察

目的 比较荧光定量ＰＣＲ与ＲＴ－ＰＣＲ（定性）检测不同检材中的ＨＣＶ－ＲＮＡ,评价荧光定量ＰＣＲ技术测定ＨＣＶ－ＲＮＡ水平的价值。方法 采用荧光定量ＰＣＲ及ＲＴ－ＰＣＲ（定性）技术同时检测了７１例血液透析患者的血清,血浆,淋巴细胞、尿液标本中ＨＣＶ－ＲＮＡ。结果 血清、淋巴细胞,尿液标本中ＨＣＶ－ＲＮＡ的荧光定量ＰＣＲ检出率（４６．３８％,８０．００％）,分别高于相应标本中ＨＣＶ－ＲＮＡ的ＲＴ－Ｐ     

Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region

The 5'-noncoding region of hepatitis C virus (HCV) genomes is highly conserved. A two-stage polymerase chain reaction (PCR), involving two pairs of primers deduced from the 5'-noncoding region of the HCV genome, was developed for a sensitive and specific detection of HCV RNA. The first stage of PCR was performed for 35 cycles with primers capable of multiplying fragments of 221 base pairs. PCR products in samples negative for HCV RNA were subjected to the second stage of PCR for 30 cycles with primers located internal to those employed in the first stage of PCR. The two-stage PCR detected up to 10 chimpanzee infectious doses/ml of HCV, and HCV RNA in 11 (92%) of 12 sera from patients with chronic non-A, non-B hepatitis without detectable antibodies to HCV by a commercial assay kit. Primers from the 5'-noncoding region of the HCV genome would be suitable for detecting HCV RNA by PCR, since the other regions of the HCV genome diverge extensively in sequence because of its nature as an RNA virus.