生物素标记HBV RNA探针的制备及应用

本文首次采用SP_(65)特殊质粒与人类的乙型肝炎病毒DNA(HBVDNA)重组。制备了Bio—HBV RNA探针,能特异地与HBV DNA杂交。并将该探针与缺口转移(Nick—translation)方法标记的Bio—HBV DNA探针进行了比较。结果显示出Bio—HBV RNA探针的敏感性比Bio—HBV DNA探针的敏感性提高10倍。本实验还分别应用了两种探针同时检测了70例乙肝病人血清中HBV DNA。阳性率各为31.42％,28.57％(P>0.25)。并对Bio—HBV RNA探针应用于临床检测乙肝病毒DNA的效能进行了初步的探讨。     

实时荧光定量PCR检测莫氏立克次体

目的 采用TaqMan-MGB探针建立检测莫氏立克次体的实时荧光定量PCR.方法 依据莫氏立克次体外膜蛋白B基因(ompB)设计引物和探针,以克隆的ompB作模板建立实时荧光定量PCR方法.结果 建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.995);该方法能检出＜10拷贝的莫氏立克次体DNA,敏感性为普通PCR的1 000倍.用该方法检测莫氏立克次体DNA,结果为阳性,但是检测其他相关细菌DNA的结果均为阴性.用该方法检测莫氏立克次体感染豚鼠血标本,50%样本检测为阳性,而普通PCR检测结果均为阴性.结论 该实时荧光定量PCR具有很高的特异性和敏感性,适合于快速检测样本中微量莫氏立克次体DNA,可用于临床实验室快速确诊地方性斑疹伤寒.     

病毒性肝炎联合诊断cDNA芯片的研制与临床应用性研究

目的 制备病毒性肝炎联合诊断cDNA芯片,并进行系列优化及临床应用性研究.方法 对于基因组较小的HBV、HDV病毒,采用Primer Premier 5.0软件针对其保守区域设计特异引物,普通PCR技术制备探针;利用限制性显示PCR(RD-PCR)技术制备基因组较大且复杂的HCV芯片探针.为了便于摸索实验条件和更好地进行探针优化,先后制备3种芯片,包括HBV与HDV诊断芯片、HCV诊断芯片及HBV、HDV、HCV联合诊断芯片.结果 杂交结果显示HBV与HDV诊断芯片、HCV诊断芯片的诊断效果较为满意.从以上2种芯片中选取探针制备HBV、HDV、HCV联合诊断芯片,并对该芯片的检测质量进行初步评估:①检测线性:病毒质粒拷贝数在104～1011copies/ml之间时,芯片检测法显示了较好的线性(r分别为0.990 2、0.992 1、0.981 9,P＜0.01);②特异性:检测黄热病病毒(YFV)、乙型脑炎病毒(JEV)、登革热病毒(DV)等其他病毒样品结果均为阴性,说明该芯片特异性较好;③重复性:检测HBV、HDV、HCV的批内精密度变异系数(Cv)值为7.1%、7.2%、6.6%,批间精密度Cv值为7.9%、8.2%、7.6%;④准确性:将HBV、HDV PCR片段(共14条)、HCV限制性片段(24条)和部分临床阳性血清(PCR产物)全部进行序列测定,在GenBank中进行Blast比较,结果表明克隆基因均属于相应基因,与理论一致.为了适应临床诊断的要求,对实验条件进行优化,包括减少杂交时间、取消预杂交、将杂交温度提高到52℃等措施.在实验室对芯片进行初步评估后,对临床血清标本进行小规模批量检测实验: 用芯片法和实时定量PCR法检测HBV阳性血清(98例)、HCV阳性血清(42例)和阴性血清(130例),对两种方法进行对比,结果显示芯片法检测与实时定量PCR法有良好的相关性(HBV、HCV的r值分别为0.985 4和0.958 2),检测HBV、HCV、HDV的敏感性和特异性分别为85.7%、76.2%、80.0%和96.7%、95.0%、100%.结论 本研究研制的联合诊断芯片敏感性、特异性均较佳,临床应用前景较好.     

DNA分子杂交法检测乙型肝炎患者血清中HBV—DNA结果分析

我们自1988年以来,采用~(32)P标记的HBV—DNA探针对634例乙型肝炎患者进行了HBV—DNA检测。现将结果报告如下。病例来源 634例乙型肝炎均为我科住院患者,经临床及实验室检查确诊的各型乙型肝炎患者。诊断标准均符合第四次全国病毒性肝炎会议制定的标准。     

用核糖体RNA ITS区序列寡核苷酸探针鉴定临床常见真菌

目的：建立一种快速鉴定临床常见真菌菌种的方法。方法:选取真菌核糖体RNA的ITS区为靶基因，设计通用引物和种特异性探针，将3个属的6种临床常见真菌(白色念珠茵、热带念珠茵、光滑念珠茵、新型隐球菌、烟曲霉和黄曲霉)的种特异性探针加尾后固定于硝酸纤维素膜上；用标记生物素的真菌通用引物扩增11种医学真菌DNA，产物与固定在膜上的探针杂交。结果：通用引物可以扩增所试11种医学重要真菌的DNA，扩增片段长度约为560bp。6种真菌扩增产物只与其对应的探针杂交。结论：此方法快速、简便、特异，适合常规实验室开展。     

乙型肝炎复制指标的相关性分析及其临床应用价值研究

 目的探讨HBeAg、HBcAg、Pres1Ag、HBcAb和HBV DNA的关系及其临床应用评价.方法Pres1Ag和HBV M检测采用酶免法,HBV DNA采用荧光探针技术定量PCR法,HBcAg采用固相放射免疫法.结果5项复制指标分别在HBV M不同模式组检出率不等同;HBcAb(+)组的HBV DNA和HBcAg阳性率显著高于HBcAb(-)的HBV M模式组(P<0.01);若以HBV DNA检测阳性为HBV复制金标准,HBcAg检测符合率(83.2%)显著高于其他指标(P<0.01),而HBcAb检测特异性和HBeAg检测灵敏度过低.结论(1)HBcAg与HBV DNA相关性最好.(2)5项指标评价HBV复制的临床意义为:HBV DNA>HBcAg>Pres1Ag>HBeAg>HBcAb.(3)HBcAb仅仅可作为HBV复制的初筛指标.     

DNA探针的高效标记和哺乳动物细胞转化子的检测

本实验采用随机引物法标记探针DNA.探针DNA加热变性成单链,以单链DNA为模板,六聚脱氧核苷酸为引物,在DNA多聚酶I大片段的作用下进行放射性标记.标记探针的比放射性达10~8cpm/μg DNA以上.放射性底物的掺入率为60%.用该方法标记的人高度重复顺序探针,可以检出微微克水平的阳性分子.与人HeLa S_3细胞DNA转化的小鼠LTK~+细胞DNA打点杂交呈阳性,与未经转化的小鼠LTK~-细胞DNA杂交为阴性,证明小鼠转化细胞中存在HeLa细胞DNA,DNA转化是成功的.     

国产和进口~(32)P标记HBV-DNA探针的应用探讨

我们用进口~(32)P标记的HBV-DNA(所用探针及方法由北医附院提供,下简称甲法)和国产~(32)P标记的HB V-DNA探针(探针及基本方法由第一军医大学提供,下简称乙法),以斑点分子杂交法对肝炎患者血清进行比较检测。结果:甲法检测124例HB_3Ag阳性者,DNA(+)80例,阳性率占64.5％;乙法检测64例HBsAg(+)者,DNA(+)40例,占62.5％。两法阳性率无差异。乙法DNA(+)的40例中,甲法同样阳性的38例,两者符合率95％。按血清中病原学指标分组其中HBsAg(+)和HBeAg(+)的74例,DNA(+)71例,占95.9％,HBsAg(+)而HBeAg(-)的40例,DNA(+)14例,占35％。     

应用Southern吸印滤膜杂交法检测HBcAg基因片段

乙型肝炎是一种由DNA病毒引起的发病率很高的传染病。为了提高乙型肝炎的诊断率,近年来出现了许多敏感的特异的血清免疫学诊断方法。作为诊断乙型肝炎用的HBcAg多从病人血清或尸肝中提取,因来源困难故不能广泛使用。最近国外有人采用遗传工程的方法由大肠杆菌合成HBcAg代替由病人血清或肝脏提取的HBcAg,亦获得满意效果。我们采用此法也获得成功。在此项研究中,为了由克隆株取得含有HBcAg基因的DNA片段,我们采用缺口翻译法将已知HBc(adw)基因片段用同位素标记制成探针,并采用Southern吸印滤膜杂交法检测五株克隆DNA酶切片段,从中找到     

白介素-Ⅱ基因片段核苷酸顺序分析

本文采用~(35)S标记的脱氧三磷酸核苷酸,以双脱氧核苷酸链终止法,分析了克隆于M13噬菌体的白介素-Ⅱ(以下简称IL-Ⅱ)DNA AluI片段的序列。其中包括XbaⅠ、HaeⅢ、B_(st)NI、HinfI限制性核酸内切酶位点。对于研究IL-Ⅱ基因重组、探针制备以及基因表达具有一定的意义。     

RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

改良裂解法提取微量组织DNA的研究

为了探讨微量组织核酸的更快速、简便、有效的提取方法,采用改良裂解法提取２５ 例微量胃粘膜组织标本的核酸,同时与经典的饱和酚抽提法和简便的消化煮沸法进行比较（ 各２５ 例） ,用１ ％ 琼脂糖电泳观察结果,并用分光光度法检测提取核酸的量。结果显示该法的提取纯度接近酚抽提法,所获核酸的量较多,而所需的时间最短;由于该法的裂解液中含有 Ｒ Ｎ Ａ 酶抑制剂,所以除了可以提取 Ｄ Ｎ Ａ,还可以获得一定量的 Ｒ Ｎ Ａ,用于反转录 Ｐ Ｃ Ｒ,对微量组织的 Ｒ Ｎ Ａ 提取提供了一种有效而实用的方法。     

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.     

Internal validation of STRmix™ for the interpretation of single source and mixed DNA profiles

The interpretation of DNA evidence can entail analysis of challenging STR typing results. Genotypes inferred from low quality or quantity specimens, or mixed DNA samples originating from multiple contributors, can result in weak or inconclusive match probabilities when a binary interpretation method and necessary thresholds (such as a stochastic threshold) are employed. Probabilistic genotyping approaches, such as fully continuous methods that incorporate empirically determined biological parameter models, enable usage of more of the profile information and reduce subjectivity in interpretation. As a result, software-based probabilistic analyses tend to produce more consistent and more informative results regarding potential contributors to DNA evidence. Studies to assess and internally validate the probabilistic genotyping software STRmix™ for casework usage at the Federal Bureau of Investigation Laboratory were conducted using lab-specific parameters and more than 300 single-source and mixed contributor profiles. Simulated forensic specimens, including constructed mixtures that included DNA from two to five donors across a broad range of template amounts and contributor proportions, were used to examine the sensitivity and specificity of the system via more than 60,000 tests comparing hundreds of known contributors and non-contributors to the specimens. Conditioned analyses, concurrent interpretation of amplification replicates, and application of an incorrect contributor number were also performed to further investigate software performance and probe the limitations of the system. In addition, the results from manual and probabilistic interpretation of both prepared and evidentiary mixtures were compared.The findings support that STRmix™ is sufficiently robust for implementation in forensic laboratories, offering numerous advantages over historical methods of DNA profile analysis and greater statistical power for the estimation of evidentiary weight, and can be used reliably in human identification testing. With few exceptions, likelihood ratio results reflected intuitively correct estimates of the weight of the genotype possibilities and known contributor genotypes. This comprehensive evaluation provides a model in accordance with SWGDAM recommendations for internal validation of a probabilistic genotyping system for DNA evidence interpretation     

A review of the collaborative exercises on DNA typing of the Spanish and Portuguese ISFH working group

Since 1992 the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Haemogenetics (ISFH) has been organizing collaborative exercises on DNA profiling with the aim of making progress on standardization and discussing technical and statistical problems in DNA analysis. A total of four exercises (GEP-92 to GEP-95) have been carried out until now. A consequence of these exercises was the creation of a quality control programme in Spain and Portugal in 1995 which was carried out simultaneously with the GEP-95 exercise. The number of participating laboratories increased from 10 in the first exercise (GEP-92) to 19 in the last exercise (GEP-95). Despite this increasing number of participating laboratories, results remained satisfactory. In the last exercises, all the laboratories used PCR-based DNA polymorphisms with an increasing number of markers obtaining good results. SLPs were used by only 30% of laboratories in the last two exercises but the results indicated a good level of expertise in most of these laboratories. The reasons for these successful results are the common use of the EDNAP protocol for SLP analysis and commercially available kits or common sequenced allelic ladders for PCR-based DNA polymorphisms. Received: 7 February 1997 / Received in revised form: 17 April 1997     

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.     

RNA/DNA co-analysis from human saliva and semen stains – Results of a third collaborative EDNAP exercise

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10–0.01 μl saliva, 5–0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.     

An optimized procedure for obtaining DNA from fired and unfired ammunition

Gun crimes are a significant problem facing law enforcement agencies. Traditional forensic examination of firearms involves comparisons of markings imparted to bullets and cartridge casings during the firing process. DNA testing of casings and cartridges may not be routinely done in crime laboratories due a variety of factors including the typically low amounts of DNA recovered. The San Diego Police Department (SDPD) Crime Laboratory conducted a study to optimize the collection and profiling of DNA from fired and unfired ammunition. The method was optimized to where interpretable DNA results were obtained for 26.1% of the total number of forensic casework evidence samples, and provided some insights into the level of secondary transfer that might be expected from this type of evidence. Briefly detailed are the results from the experimental study and the forensic casework analysis using the optimized process. Mixtures (samples having more DNA types than the loader’s known genotype detected or visible at any marker) were obtained in 39.8% of research samples and the likely source of DNA mixtures is discussed.     

Standardization of 152Eu.

152Eu was standardized within the frame of a BIPM international comparison. The solution was prepared and bottled by PTB. The ampoules were measured at BIPM in the SIR ionization chambers and then dispatched to the participating laboratories. In the Radionuclide Metrology Laboratory of IFIN-HH, the solution was standardized using a coincidence method with "beta-efficiency extrapolation" by foil absorption. A simple 4pi beta-gamma-coincidence system was used, with NaI(Tl) and 4piPC detectors. No significant impurities were found apart for 0.5% 154Eu. To obtain linear extrapolation plots, a "gamma-window" of 180-1500 keV was chosen. Dead-times of 10 micros were used. A combined uncertainty of 0.24% was obtained, mainly from the extrapolation procedure component.     

Results of the intercalibration study of laboratories involved in assessing the environmental consequences of the Chernobyl accident

Within the framework of the International Chernobyl Project, the IAEA's Seibersdorf Laboratories organized an intercalibration exercise among some of the laboratories which were involved in assessing the environmental contamination in the USSR due to the accident. The objective was to assess the reliability of the radioanalytical data for food and environmental samples, which wre used to assess the doses. In the initial study reference materials from the stocks of the IAEA's Analytical Quality Control Services (AQCS) were re-labelled and submitted to 71 laboratories as blind samples. These natural matrix materials included samples of milk (containing 2 different levels of radioactivity), soil, air filters and clover. The concentrations of radionuclides in these samples were known from previous intercalibration exercises.The overall range in performance was broad, which is similar to what has been observed in previous international intercomparisons. The results obtained by gamma-ray spectrometry tended to be somewhat underestimated, on average. On the other hand, the laboratories showed an overall tendency to overestimate ⁹⁰Sr and possibly ²³⁹Pu, which were analysed radiochemically.The intercalibration exercise is continuing with nine materials, including: soil, grass, hay and milk powder contaminated with fallout from the Chernobyl accident. These materials, which were prepared by laboratories in the USSR, are now being tested by AQCS prior to future intercomparison exercises. Work with these materials is expected to continue for several years.