磁珠法结合定量PCR检测肠道病毒71型灭活疫苗中宿主细胞DNA残留量的验证及应用

目的  对磁珠法结合定量PCR（quantitative PCR,qPCR）检测肠道病毒71型灭活疫苗〔非洲绿猴肾细胞（Vero细胞）〕（EV71疫苗）中宿主DNA残留量进行适用性验证及应用。方法  利用微磁珠与蛋白溶液中DNA的特异性结合,来提取样品中残留的Vero细胞DNA。将已知浓度Vero细胞DNA对照系列稀释后,作为标准品DNA与提取的DNA同时进行qPCR扩增。根据标准品DNA的循环阈值与浓度之间的线性关系,对未知样品中残留DNA进行定量分析。结果  标准品检测范围在0.03～3 000.00 pg/反应。该方法的标准曲线决定系数≥0.98,扩增效率在90%～110%。质控样品回收率在50%～150%,相对标准偏差均小于30%。实验结果的各项参数均在要求范围内。结论  磁珠法可解决残留DNA检测中样品前处理的技术难点,qPCR能够简便、快速、准确地对生产过程样品的EV71疫苗中DNA残留量进行定量测定。适用于EV71疫苗中DNA残留量的检测及疫苗生产过程和其成品的质量控制,对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。     

Sabin株脊髓灰质炎灭活疫苗Vero细胞DNA残留定量PCR检测法的验证

目的  验证Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留定量PCR（quantitative PCR,qPCR）检测法。方法  用试剂盒提取Sabin株脊髓灰质炎灭活疫苗原液的DNA,采用qPCR法检测Vero细胞DNA,并验证方法的线性、专属性、准确性、精密度、耐用性及定量限;同时采用DNA探针杂交法检测样品。结果  qPCR检测Vero细胞DNA标准曲线在0.003 0~300.000 pg/μl之间线性良好;对Hep-2细胞、大肠埃希菌、酵母细胞DNA均无扩增反应;高、中、低浓度样品加标回收率在50%~150%,准确性良好;定量检出限为0.003 0 pg/μl;精密度良好（相对标准偏差＜15%）;反应体系配制好后置于2~8 ℃ 30 min后进行检测,相对标准偏差＜15%,耐用性良好。DNA探针杂交法与qPCR结果均小于0.1 pg/μl。 结论  qPCR检测Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留具有良好的线性、专属性、准确性、精密度和耐用性,可适用于该项检测。     

芋螺基因组DNA提取方法的优化

热带药用海洋生物芋螺产生的芋螺毒素(Conotoxin),已成为神经科学研究的有力工具药和新药开发的新来源。近来出现了从芋螺基因组DNA中克隆新型芋螺毒素基因的新方法．因而快速获得芋螺基因组DNA是分离多样性毒素基因、建立基因库及药用芋螺基因资源开发的前提。本研究采用不同的DNA提取方法．从6种芋螺的不同组织和器官中分离总DNA，经综合比较，建立了简便快速的提取芋螺总DNA的改良苯酚-SDS优化方法。     

荧光定量PCR法测定地特胰岛素原料中酿酒酵母宿主DNA残留量

目的建立检测地特胰岛素原料中宿主DNA残留量的荧光定量核酸扩增(Real-time PCR)方法并进行验证,用于该产品的质量控制。方法选择酿酒酵母5s RNA作为靶基因设计引物,提取地特胰岛素原料中的残留宿主DNA,采用Real-time PCR SYBRGreen染料法对标准DNA和样品进行测定,绘制标准曲线并分析样品中的DNA残留量。对建立的方法进行方法学验证,并测定3批地特胰岛素原料中的残留宿主DNA。结果酿酒酵母基因组DNA质量浓度在0.18～180000ng/mL线性良好(r~2=0.998 5);回收率均在80.0%～106.3%,检测3批地特胰岛素原料的宿主DNA残留量均低于进口药品注册标准限度。结论该方法可用于酿酒酵母生产的地特胰岛素原料中残留宿主DNA的定量测定。     

实时荧光定量PCR法检测乙型肝炎患者血清HBVDNA含量512例分析

目的探讨实时荧光定量PCR检测不同血清标志物模式的乙型肝炎患者的血清HBVDNA含量的特点。方法应用ELISA对512例病人血清中乙型肝炎血清标志物进行检测,同时用实时荧光定量PCR检测标本中HBVDNA的含量,分别统计每组的HBVDNA阳性例数及其阳性率,用u检验进行统计学处理。结果HBsAg（＋）／HBeAg（＋）／抗HBc㈩组168例,HBVDNA阳性161例,占95．8％;HBsAg（＋）／抗HBe（＋）／抗HBc（＋）组163例,HBVDNA阳性51例,占31．3％;HBsAg（＋）／抗HBc（＋）组181例,HBVDNA阳性94例占51．9％;三组之间互相比较差异均有显著性（P〈0．001）。结论HBsAg（＋）／HBeAg（＋）／抗HBc（＋）组中HBV复制最活跃,实时荧光定量PCR检测HBVDNA比血清标志更能反映乙型肝炎病毒存在与复制情况。     

A new quantitative PCR assay for the detection of hepatotoxigenic cyanobacteria

Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml⁻¹. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA及其与血清标志物的关系探讨

目的　用荧光定量聚合酶链反应 (fluorescence quantitative polymerase chain reaction,FQ- PCR)方法定量检测血清中乙型肝炎病毒 (hepatitis B virus,HBV)的数量及探讨其与 HBV标志物 (HBV M)表现模式的关系 ,以指导临床。方法　共 2 4 4份临床血清标本 ,HBV DNA定量采用荧光定量 PCR分析系统 ,HBV M采用 EL ISA法。结果　经 FQ- PCR检测 ,99例 HBs Ag(+) / HBe Ag(+) / HBc Ab(+)的标本 ,其血清标本 HBV DNA亦全部阳性 ,平均 HBV DNA拷贝数为 1.82× 10 7copies/ ml;96例 HBs Ag (+) / HBe Ab (+) / HBc Ab (+)的标本 ,其 HBV DNA检出率为 6 6 .7% (6 4例 ) ,平均拷贝数为 1.82× 10 4 copies/ m l;11例 HBs Ag (+) / HBc Ab (+)的标本其 HBV DNA检出率为 6 3.6 % (7例 ) ,平均拷贝数为 7.94× 10 4 copies/ ml。结论　血清 HBV DNA水平与 HBV M表现模式有关 ,HBs Ag (+) / HBe Ag (+) / HBc Ab (+)的标本 HBV DHA值显著高于 HBs Ag (+) / HBe Ab (+) / HBc Ab (+)的标本和 HBs Ag (+) / HBc Ab (+)的标本 ,提示 HBs Ag与 HBe Ag的存在影响 HBV DNA水平。 FQ- PCR能实现准确定量 ,可以检测 HBV的真实感染和复制情况 ,对于乙型肝炎的临床诊断、治疗方案的选择和疗效考察有较大的指导意义     

Development and characterization of a novel host cell DNA assay using ultra-sensitive fluorescent nucleic acid stain “PicoGreen”

Development of a novel host cell DNA assay using PicoGreen is described, which is capable of detecting short double stranded DNAs (ds-DNAs) in cell culture supernatants and process intermediates. Examination of this PicoGreen DNA assay was carried out by determination of the DNA length detection limit, observation of short ds-DNAs in cell culture supernatants and process intermediates, evaluation of dose dependency and a supersensitizing protocol, and comparison of the novel assay with conventional assays for measuring host cell DNA concentration in real samples. The PicoGreen DNA assay was capable of detecting ds-DNAs as short as 20 bp, and the sensitivity of the PicoGreen DNA assay was comparable to that of the Threshold system with application of additional SDS/Proteinase K digesting and DNA concentrating steps. Also, the amount of DNA identified in both cell culture supernatant and process intermediates was clearly underestimated by the Threshold system results when compared with the PicoGreen DNA assay results. The PicoGreen DNA assay clearly provides better accuracy and is a simpler procedure for measuring host cell DNA levels in cell culture supernatant and process intermediates than the conventional method with the Threshold system. This newly developed DNA assay will be prominent among host cell DNA assays for measuring host cell DNA levels in bio-pharmaceuticals.     

实时荧光定量PCR法检测食管癌组织中细胞周期蛋白CyclinD1和CyclinA基因的表达

目的检测食管癌组织中细胞周期蛋白CyclinD1和CyclinAmRNA的表达水平，探讨相关基因表达与食管癌发生、发展的相关性。方法应用SYBRGreen染料法进行相对实时荧光定量PCR检测CyclinD1和CyclinA在48例食管癌组织及其癌旁正常食管组织中的表达情况。结果CyclinD1和CyclinA在食管癌组织中的表达水平均明显高于正常食管黏膜组织，差异具有统计学意义（P〈0．05）。结论人食管癌组织中CyclinD1和CyclinA的高表达可能与食管癌的发生发展密切相关，有望作为食管癌发生发展的评估指标。     

实时定量PCR法测定乙肝病毒脱氧核糖核酸

目的：建立实时定量PCR法测定血清样品中乙肝病毒脱氧核糖核酸（HBV—DNA）含量。方法：用PEG-裂解-煮沸法从血清中提取核酸,制作成标准浓度曲线后,对100份患者样本进行实时荧光定量分析,并与ELISA（酶标法）结果作比较,再与CAP—CTM法（化学发光法）进行比较。结果：用SPSS（V．16）软件进行统计分析,得线性回归方程y=-3．212X＋44．90;r^2=0．9980;日内、日间平均误差为RSD〈2．15％：最低检测限是400copies／mL（拷贝数）：该法测得患者HBV—DNA的浓度与EUSA测出样品阳性发生率呈正相关,再与CAP—CTM作配对t检验,P〉0．05,两者无显著性差异。结论：新建的PCR检测法敏感度高,重现性好,线性范围较广,可用于进行临床血清样本中HBV—DNA的定量检测,这对评估HBV患者诊治效果有帮助。     

Development of the species-specific multiplex PCR and DNA sequencing methods for rapid authentication of Isatidis Folium and its adulterants

Isatis indigotica Fort. (family Cruciferae), is an herb widely used in traditional herbal medicine and its dried leave was named “ISATIDIS FOLIUM”. Baphicacanthus cusia (Ness) Bremek. and Polygonum tinctorium Ait. are commonly misused as ISATIDIS FOLIUM in Chinese Medicine pharmacy. For the purpose of being not misused, specific primers based on the sequence difference of chloroplast trnH-psbA intergenic spacer were designed and multiplex polymerase chain reaction method (multiplex PCR) was developed. In this study, 29 original herbal materials were analyzed and our results show that DNA size after multiplex PCR was able to distinguish variations between three herbs. DNA fragments of 464, 297, 170 base pairs (bps) were represented for I. indigotica and B. cusia and P. tinctorium, respectively. In conclusion, our investigations demonstrate that molecular identification method provides more accurate results for medicinal plants detection and good quality control of ISATIDIS FOLIUM.     

SYBR-Green实时定量PCR用于Vero宿主细胞DNA残留量的检测

目的:建立基于SYBR-Green荧光染料实时定量PCR检测Vero宿主细胞DNA残留量的方法。方法:提取Vero细胞基因组DNA,设计细胞基因组高度重复顺序AGMr(HindIII)-1基因片段的特异性引物,通过PCR扩增AGMr(HindIII)-1序列中一段特异性cDNA片段,克隆至pGEM-T Vector,重组质粒经酶切及测序鉴定合格后命名为pGEM-T/AGMr(HindIII)-1-S。结果:使用重组质粒和细胞基因组DNA分别作为检测标准品,均取得了良好的结果,其检测灵敏度分别达到了0.03 fg.μL-1和0.03 pg.μL-1。结论:SYBR-Green实时定量PCR可用于Vero宿主细胞DNA残留量的准确定量。     

生物制品中宿主细胞残留DNA检测的研究进展

宿主细胞残留DNA是指可能出现于生物制品中的来自宿主细胞的DNA片段。用传代细胞株生产的生物制品,其宿主细胞残留DNA可能会传递肿瘤或病毒相关基因,存在潜在的危险性。各国药品监督管理机构对宿主细胞残留DNA的残留量有着严格的限度控制,同时各国药典也提供数种经典的检测方法。建立合适的宿主细胞残留DNA检测方法有助于监测生产工艺,确保生物制品的安全性和质量。此文对宿主细胞残留DNA潜在的危害、国内外监管机构对宿主细胞残留DNA检测标准和检测方法以及各方法的特点及研究进展做一综述。     

Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water

Giardia lamblia is one of the most recognized waterborne protozoan parasites causing gastrointestinal disease. A simple but effective DNA extraction protocol for real-time PCR detection from surface water samples was developed in this study. Eleven protocols were compared, which consisted of freeze–thaw treatments (liquid N₂ and boiling water) and purification using the Qiagen DNeasy kit, together with different combinations of proteinase K, PVP360, GITC and Chelex 100 incubation. Using concentrated surface water samples spiked with G. lamblia cysts, the necessary steps for high DNA recovery were shown to be freeze–thaw, DNeasy purification and Chelex 100 incubation. Multiple rounds of freeze–thaw treatment (five cycles per round) were reported for the first time in this study to significantly increase the DNA yield from G. lamblia cysts, from ~20% after one round of freeze–thaw to 40 and 70% after two and three-rounds of freeze–thaw, respectively. More than three rounds of freeze–thaw treatment did not promote additional DNA recovery. The final protocol included three–three-rounds of freeze–thaw treatment, DNeasy purification and Chelex 100 incubation. This method was simpler, more cost-effective, and had a comparable DNA recovery to methods involving immunomagnetic separation.     

Reliability of non-invasively acquired human genomic DNA as a substrate for real-time PCR-assisted analysis of genetic polymorphisms

Molecular epidemiological studies require high numbers of participants. The combination of an non-invasive access to human DNA with a rapid genotyping analysis, e.g. by use of LightCycler assisted real-time polymerase chain reaction (PCR), can be helpful in conducting such trials. The aim of our study was to define, for the first time, the use of LightCycler technology in analysis of non-invasively derived DNA. DNA extracted from blood, mouthwash and buccal cytobrush samples from 100 volunteers was analyzed for the genotypes of cytochrome P450 CYP1B1, and glutathione S-transferases GSTT1, GSTM1 and GSTP1. The median amounts of DNA isolated from blood, mouthwash and buccal cytobrush samples were 95, 11 and 8 µg, respectively. While genotyping for CYP1B1 codon 432 polymorphism and GSTP1 codon 105 polymorphism resulted in a complete correspondence for all three modes of sampling, the identification of individuals with null-genotype for GSTT1 or GSTM1 failed in some cases due to atypical courses of the corresponding melting curves, leading to high false-positive rates in the group of non-invasively derived samples. Thus, the results presented here call for caution in using LightCycler assisted real-time PCR in non-invasively collected samples, at least when appropriate control strategies are not implemented.T. Neuhaus and G. Geisen contributed equally to this work     

荧光定量PCR法检测鼻咽癌血浆游离EBV DNA的临床意义

目的通过检测血浆中游离EBV DNA水平,探讨其在鼻咽癌诊断、鉴别诊断、临床分期中的临床意义,方法采用荧光定量PCR方法定量检测111例初治鼻咽癌患者和107例放疗后持续缓解患者血浆游离EBC DNA水平．结果初治组及临床缓解组血浆中游离EBV DNA检出率分别为74．8％（83／111）和4．6％（5／107）．初治组检出率显著高于临床缓解组．在统计学上有显著性差异;初治组中晚期（Ⅲ＋Ⅳ期）血浆游离EBV DNA拷贝数显著高于早期（Ⅰ＋Ⅱ期）;N晚期（N2＋N3）拷贝数显著高于早期（N0＋N1）;T晚期（T3＋T4）拷贝数显著高于早期（T1＋T2）;统计学上均有显著性差异。结论荧光定量PCR方法检测血浆中游离EBV DNA是一种敏感可靠的方法,血浆游离EBV DNA定量检测对鼻咽癌诊断、鉴别诊断、分期、治疗后监测有重要的临床意义．有望成为鼻咽癌肿瘤学标记物。     

实时荧光PCR法检测脑出血患者ApoE基因多态性的意义及探讨

目的探讨载脂蛋白E（ApoE）基因多态性与脑出血的关系及实时荧光聚合酶链反应（PCR）技术在ApoE基因多态性检测中的应用价值。方法对福建附一医院住院的74例脑出血患者进行研究,正常对照组为80例本院健康体检者;ApoE基因型采用实时荧光PCR方法检测,血清TC、TG、LDL、HDL在全自动生化仪上采用动力学法检测。结果脑出血患者组ApoEε4等位基因频率为14.9%,明显高于正常对照组（P〈0.01）;比较不同ApoE基因型脑出血患者的血清TC、TG、LDL、HDL含量,结果ε4组的TC含量明显高于ε3和ε2组（P〈0.05,P〈0.01）,LDL含量明显高于ε3和ε2组（P〈0.05）。结论ApoEε4等位基因可能是脑出血的易感因子和风险因素;实时荧光PCR技术检测ApoE基因多态性具有方便、快捷的优点,值得推广。     

炭疽杆菌双重实时荧光PCR检测方法的建立

目的 建立同时检测炭疽杆菌capA基因、PA基因的双重实时定量荧光PCR方法,用于应对突发传染病疫情和流行病学调查、突发公共卫生事件应急处置的早期检测及防范生物恐怖威胁.方法 设计和合成分别针对炭疽杆菌capA基因、PA基因的引物对和荧光双标记探针,构建质粒标准品,通过优化探针、引物浓度、反应体系试剂组分等参数,建立可同时检测capA基因、PA基因的双重实时荧光PCR方法,测试方法的灵敏度和特异性,并在实战检测中应用.结果 双重实时荧光定量PCR的炭疽杆菌capA基因、PA基因检测的灵敏度分别可达到每反应5和50拷贝,并具有良好的特异性,在实战应用中亦经过考验.结论 双重实时荧光定量PCR技术具有可以同时筛查、经济、快速、特异性强等优点,在炭疽杆菌检测方面有良好应用价值.     

定量PCR法和DNA杂交法检测聚乙二醇修饰尿酸酶原液中DNA残留量的比较

目的 建立快速准确的聚乙二醇修饰重组尿酸酶(PEGylated uricase,PEG-UHC)原液DNA残留量测定法,为其他多位点PEG修饰蛋白药物的DNA残留量检测提供参考。方法 参照中国药典2020年版三部“外源性DNA残留量检测法”第一法DNA探针杂交法和第三法定量PCR法,检测PEG-UHC原液DNA残留量;对定量PCR法进行检测限度、线性范围、准确度和精密度等方法学验证;进行3批次PEG-UHC原液DNA残留量测定。结果 探针杂交法DNA残留量测定线性范围为1×10^-4~0.1 ng·µL^-1,3批次PEG-UHC原液未见明显显色,DNA残留量均低于每剂10 ng。PCR法检测限度为1×10^-5 ng·µL^-1,DNA浓度在1×10^-4~100 ng·µL^-1内线性关系良好(R²=0.999)。不同浓度的标准DNA在PEG-UHC原液中的回收率范围为92.57%~114.17%,表明PEG偶联物对定量PCR影响较小。3批次PEG-UHC原液中DNA残留量分别为每剂0.143,0.187,0.154 ng,符合国家药品监督管理局限量要求。结论 DNA探针杂交法为传统定性检测,耗时较长,准确度较低。定量PCR法操作简便快速、灵敏度高,且可定量分析,适于PEG-UHC等多位点PEG修饰蛋白药物DNA残留量测定。     

丝状真菌顶头孢霉染色体DNA的提取

目的探索提取头孢菌素C产生菌顶头孢霉基因组DNA的方法。方法采用氯化苄法、液氮研磨法和酶裂解法。结果较其他两种方法比 ,氯化苄法提取效果好。当提取液pH值为 9 0～10 0、EDTA浓度为 12 0mmol·L-1时 ,所得的顶头孢霉染色体DNA的质量和数量均较理想 ,每克(湿重 )顶头孢霉菌丝体能得到 96 μg的染色体DNA。常规琼脂糖凝胶电泳分析 ,其DNA片断大于2 3kb。结论该分离方法获得的染色体DNA质量较高 ,可进行限制性内切酶酶解并适合后续特定基因的PCR扩增反应