分子杂交法研究肝炎病人血清和肝组织中输血传播病毒

目的 对各型肝炎病人血清和肝组织中输血传播病毒（TTV）核酸检测分析,探讨病毒的致病性。方法 以地高辛为标记物制备TTV DNA探针,斑点杂交法、原位杂交法分别检测血清中及肝组织中TTV DNA。结果 检测103例血清,TTV总阳性率为25．24％（26／103）;甲－戊型肝炎组检出率21．81％（12／55）、非甲－非庚型患者检出率47．37％（9／19）,显著高于正常对照组15％（3／20）。临床可见TTV的单独感染和重叠感染;出现急性、慢性甚至重度肝损伤。12 肝组织可见TTV阳性,阳性颗粒主要见于肝细胞核内。结论 从血清和肝组织证实了TTV的存在,提示这种病毒可能是导致肝脏炎症的一种新病原。     

TTV酶联免疫方法的建立及其初步应用

目的 建立检测TTV的酶免疫技术,了解不同型肝炎患者血清中抗－TTV抗体的分布情况,并结合TTV DNA的检测分析两者的关系。方法 以原核表达的TTV ORF1蛋白为抗原建立检测抗－TTV的酶联免疫吸附试验（ELISA）方法,采用该方法检测不同肝炎患者中抗－TTV抗体;在套式聚合酶链反应（PCR）方法检测血清标本中TTV DNA。结果 所建立的检测TTV的ELISA方法具有较好的特异性,不同别肝炎患者中抗－TTV抗体阳性率分别为：甲型肝炎患者10.5%（4／38）,乙型肝炎患者12.5%（16／128）,丙型肝炎患者8.3%(7/84),丁型肝炎患者7.7%(30/93),健康人群1.3%(1/78)。统计学分析表明,非甲－庚型肝炎患者抗－TTV阳性率显著高于其他型肝炎患者（P＜0.01）,而正常人群则显著低于肝炎患者（P＜0.05）。抗－TTV阳性率与TTV DNA阳性率存在相关性（P＜0.05）。结论 在不同型肝炎患者中均可检出抗-TTV抗体,但以非甲－庚型肝炎患者阳性率高;TTV抗体可与基因同时存在于患者血清中,抗－TTV抗体可能类似抗－HCV, 是一传染性标志。     

南京市部分幼儿TT病毒DNA检测及基因序列分析

目的 了解幼儿人群中TT病毒的流行率及基因判别。方法 设计、合成引物,采用半套式聚合酶链反应（hemi-nested PCR)方法检测幼儿血清标本并进行序列分析。结果 在110份幼儿血清中,TTV DNA总检出率为12．73％。在107名ALT正常,抗HAV－IgM、HBsAg、抗HCV阴性的幼儿血清中,TTV DNA检出率为11．2％（12／107）;从1名ALT升高幼儿血清中检出TTV DNA,从2名HBsSg阳性幼儿血清中检出TTV DNA阳性血清1份。对2株TTV DNA阳性株序列分析结果显示,它们与日本分离株N22、TA278（G1a)、TX011（G1b)、TS003(G2a)、NA004（G2b)和中国株CHN1相应位置核苷酸序列的同源性分别为83．3％／86．0％、84．2％／87．8％、93．7％／98．6％、67．6％／67．1％、64．9％／64．4％、83．8％／88．3％,经系统发育分析它们属于G1b型。结论 幼儿人群中存在TTV感染,TTV流行率为12．73％。TTV感染可能存在血源传播途径外的其他传播途径,并存在健康携带状态。     

贵州地区不同人群TTV核酸检测及部分核苷酸序列分析

目的 了解贵州地区TTV感染状况，分析TTV贵州株的基因特点。方法 以公布的TTV第1读码区序列设计两对寡核苷酸引物，用套式聚合酶链反应法(nested—PCR)检测贵州地区不同人群血清中TTV核酸(TTV DNA)，并对3份TTV DNA阳性血清的PcR产物，用直接测序法测定核苷酸序列。结果 62例正常人，37例志愿献血员，50例血液透析患者，107例静脉药瘾者及139例肝病患者血清中TTV DNA阳性率分别为6．45％(4／62)，8．1％(3／37)，26.0％(13／50)，25.23％(27／107)和16.55％(23／139)。在肝病组中，重型肝炎、肝硬化、肝癌患者的TTV DNA阳性率分别为35.71％(5／14)，14．15％(15／106)和15．79(3／19)。测定的282个核苷酸中，3株贵州株的同源性均高于99％，与日本株N22相比，同源性都为98％。结论 贵州存在TTV感染，血透患者中有较高的TTV感染率，TTV可能是重型肝炎的病原因子，3株贵州株可能为同一基因型。     

石蜡包埋组织DNA提取方法的比较及巢式PCR技术的应用

确定从福尔马林固定、石蜡包埋(FFPE)组织中提取DNA的最佳方法,增强长片段PCR产物的成功扩增率,便于分子生物学与临床医学的有机结合。本文选取正常甲状腺FFPE标本20份,分别应用一步法、酚/氯仿抽提法和试剂盒法三种不同的方法提取总DNA,并应用传统PCR法和巢式PCR法对线粒体DNA(mtDNA)的D环区进行扩增。结果酚/氯仿抽提法所得样本DNA的OD260/D280比值最高,为1.703±0.086。一步法、酚/氯仿抽提法和试剂盒法提取DNA的普通PCR长片段扩增成功率分别为0%、5%和10%,而巢式PCR长片段扩增成功率分别为0%、95%和85%。可见用蛋白酶K消化、酚/氯仿纯化法提取的FFPE组织标本DNA质量可靠,巢式PCR技术是从石蜡标本中扩增长片段DNA的有效方法。     

原位杂交法检测非甲～庚型肝炎患者肝组织中TT病毒DNA

目的 证实在非甲－庚型病毒性肝炎患者肝组织中TT病毒（transfusion-transmitted virus,TTV)的存在。方法 采用地高辛素标记TTV DNA探针以原位杂交技术对51例血清学病毒标记非甲－戊型、免疫组化检测肝组织中HBsAg、HCV NS3Ag及HGV N55Aag阴性的病毒性肝炎患者石蜡包埋肝组织进行了检测。结果 各型病毒性肝炎肝组织中TTV基因的总检出率为27．5％,其中急性轻型肝炎的检出率为30．8％（4 ／13）,急性重型肝炎为1／8,亚急性重型肝炎为3／7,慢性肝炎为2／6,活动性肝硬化为2／9,慢性重肝肝炎为1／4,原发性肝癌为1／4。TTV DNA表达于肝细胞核或胞质内,以核型多见。在急性肝炎,TTV阳性细胞弥漫分布于肝小叶内,慢性肝炎于汇管区附近较为密集,而在肝硬化病例,阳性细胞在假小叶内多呈片族状不规则分布。结论 在不明原因病毒性肝炎患者血清及肝组织中TTV DNA的检出表明TTV为一种新型的肝炎病毒,TTV为一种嗜肝性病毒。     

使用PCR—SSP方法检测HLA—DR抗原

目的　采用顺序特异引物聚合酶链反应 (PCR -SSP)建立人类白细胞抗原DR位点的DNA分型方法 .方法　合成 2 9个特异性引物和 1对阳性对照引物 ,组成 2 0个PCR反应用于DR位点 ,建立一步法PCR -SSP .结果　所有样本PCR -SSP基因分型获得成功 ,分型结果经标准DNA ,限制性核酸内切酶分析证实符合 ,特异性和重复性 10 0 % .结论　PCR -SSP检测HLA -DR的方法具有快速、准确、特异性高等优点 ,适合临床应用 .     

Taqman荧光定量RT—PCR在呼吸道感染患者临床标本非SARS冠状病毒检测中的应用

目的建立检测四种常见人非SARS冠状病毒核酸特异的快速、敏感的TaqManqRT—PCR检测方法,应用于急性呼吸道感染患儿的感染分析。方法分别应用TaqManqRT—PCR与普通RT—PCR平行检测248份呼吸道标本,对方法的灵敏性、特异性和稳定性以及临床标本的适用性进行比较评价,阳性标本以体外转录RNA为标准品进行病毒载量定量。结果本方法可对HKU1、NL63、229E、OC43四种冠状病毒进行特异性诊断,与其他病毒无交叉反应,检测灵敏度可达10拷贝／μl,检测线性范围可达10^1～10^8拷贝／μl,248份标本中HKU1、NL63、229E、OC43阳性率依次为1．2％,0．8％,1．2％,1．6％,其中OC43荧光RT—PCR法检出率高于普通RT—PCR,其余三种病毒两种方法检测结果一致。非SARS冠状病毒阳性标本均检出于12月至次年5月。结论建立的TaqManRealtimeRT-PCR法具有特异性强、灵敏性高的特点,是开展非SARS冠状病毒的临床检测与疾病监测的有效技术手段。     

锁核酸捕获TaqMan探针实时聚合酶链反应构建及检测乙型肝炎病毒DNA

建立和检测新型乙型肝炎病毒(HBV)DNA检测技术锁核酸(LNA)捕获TaqMan探针实时聚合酶链反应(PCR)。方法 2009年8月至2010年3月选取本院20例健康献血者和肝病专科住院的75例经COBAS Amplicor检测HBV DNA阳性慢性乙型肝炎(CHB)患者,分别采集血液样本进行LNA捕获TaqMan探针实时PCR特异性试验。同时对LNA-实时PCR方法的重复性、特异性和线性范围等进行分析。结果 其线性范围为10～2.3×109 IU/ml。将10 IU/ml作为检测下限,具有95％可信区间。线性回归分析显示其斜率接近1.0 (R2=0.999)。批内变异值为3.88％～4.36％,批间变异值为8.5％～11.7％。20例健康献血者的阴性对照血清HBV-DNA检测均为阴性,而75例经COBAS Amplicor检测HBV DNA阳性患者的血清样本除1例阴性外,其余都为阳性。结论 LNA捕获TaqMan探针实时PCR方法为HBV-DNA检测的一种快捷灵敏、准确度高的新方法,值得临床实验室推广应用。     

TORCH与优生：Ⅲ用聚合酶链反应结合地高辛分子杂交产前诊断弓形虫感染

根据本实验室筛选出的弓形虫(ZS2株)特异DNA克隆片段的部分顺序分析的数据，设计并合成特异的寡核苷酸引物对，建立多聚酶链反应(PCR)诊断弓形虫感染的方法。不同来源弓形虫株和阳性标本DNA的PCR产物经电泳检测，均出现特异的扩增片段。以地高辛标记的该PCR产物中弓形虫特异顺序的寡核苷酸为探针，对扩增产物进行斑点杂交分析，该探针能与阳性病例的扩增产物杂交，而不与阴性病例的扩增产物杂交。用PCR结合地高辛分子杂交方法对不良生育史孕妇进行产前诊断，34例外周血白细胞DNA检测，2例阳性，分别为出生水肿胎儿和死胎；76例羊水细胞DNA检测，3例阳性，其中2例出生为无脑儿；30例绒毛DNA检测，4例阳性，均为难免流产。PCR产物结合地高辛分子杂交方法可测出少至10fg（10^-1g)的弓形虫DNA，本方法更加特异敏感。     

TT病毒在谷丙转氨酶升高的体检者和肝病患者中检 …

目的 通过研究ＴＴ病毒在谷丙转氨酶升高的体检人群和肝病患者中的意义。探讨其致病性。方法 收集１９例谷丙转氨酶升高体验者和４１例转氨酶正常的随机对照的血清,以及１８２例肝病患者的血清,采用ＰＣＲ方法检测ＴＴ病毒的ＤＮＡ。聚合酶链反应（ＰＣＲ）产物经限制性片段长度多态性（ＲＦＬＰ）分析验证。同时检测甲,乙,丙,戊,庚型肝炎病毒（ＨＡＶ,ＨＢＶ,ＨＣＶ和ＨＧＶ）感染标志。结果 １９例转氨酶升高体检者中,     

Detection of a novel DNA virus (TTV) sequence in peripheral blood mononuclear cells.

DNA sequences of a novel DNA virus (TTV) were examined in 81 peripheral blood mononuclear cell (PBMC) DNA samples from 48 children and 33 adults, 22 cord blood mononuclear cells (CBMC) DNA samples, and 7 autopsy liver tissue DNA samples by a hemi-nested polymerase chain reaction (PCR). The PCR was carried out using the published primers (NG059, NG061, NG063) to amplify TTV DNA sequences. The sequences were detected in 4 of 81 (5%) PBMC DNA samples, in none of 22 (0%) CBMC DNA samples, and in 2 of 7 (29%) liver tissue DNA samples by direct gel analysis. The PCR-amplified products were confirmed by direct sequencing. The sequencing showed considerable diversities, with differences of 0-55% in 6 TTV isolates, compared with the prototype sequence of TTV. These results suggest that TTV is a ubiquitous virus that produces asymptomatic infection in a large proportion of the general population without transfusion of blood-derived products. To our knowledge, this is the first report describing the detection of TTV DNA sequences in PBMCs.     

Detection of transfusion transmitted virus DNA by real-time PCR.

BACKGROUND: Little is known about the pathogenic role and the endemic situation of transfusion transmitted virus (TTV). OBJECTIVES: In this study, a molecular assay for detection of TTV based on automated nucleic acid extraction and real-time PCR was developed and evaluated. The new assay includes an internal control. STUDY DESIGN: After optimization of the molecular assay, 103 clinical samples were studied retrospectively. All sera had been tested for anti-HCV and anti-HIV-1 antibodies earlier. RESULTS: The amplification efficiency was found to be 102%. When clinical specimens were tested, 79 of 103 serum samples were found to be positive for TTV. There was no significant difference between various groups of patients. The internal control was detected in all negative and weak positive samples. CONCLUSIONS: This molecular assay proved to be suitable for routine detection of TTV in clinical samples. Moreover, a relative statement on the TTV serum load can be done.     

献血员中TT病毒DNA检测及部分基因序列分析

目的探讨献血员中一种与输血后肝炎有关的病毒—ＴＴＶ的感染情况。方法采用ＴＴＶ基因组ＯＲＦ１区的套式聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）方法检测２６２份献血员血清标本。结果在血清丙氨酸转氨酶（ＡＬＴ）异常、ＨＢｓＡｇ及抗－ＨＣＶ阴性的５８份献血员血清标本中，１８份ＴＴＶＤＮＡ阳性，ＴＴＶＤＮＡ的阳性检出率为３１０％；在２０４份正常献血员血清标本中检出３０份（１４７％）ＴＴＶＤＮＡ阳性，ＴＴＶＤＮＡ的阳性检出率明显低于ＡＬＴ异常献血员人群（χ２＝３８１，Ｐ＜００５）。序列分析结果显示，其序列与日本株和中国株相应位置核苷酸序列的同源性大于９７％，可能属同一基因型。结论提示我国献血员中存在ＴＴＶ感染，ＴＴＶ存在“健康携带状态”，输血可能成为传播ＴＴＶ感染的途径之一     

Indirect evidence of TTV replication in bone marrow cells, but not in hepatocytes, of a subacute hepatitis/aplastic anemia patient

The presence of a new DNA virus (TTV) has been reported in sera from patients with posttransfusion hepatitis of unknown etiology. The precise replication site of TTV, however, has not been established. In this study, the presence of TTV in liver autopsy material, and in bone marrow biopsy and autopsy samples taken from a subacute hepatitis/aplastic anemia patient was determined by PCR and Southern blot analyses. Liver cells were found to contain only TTV DNA and not mRNA. Bone marrow material, especially that taken at biopsy, contained high levels of TTV DNA. It is suggested that the TTV replication site was in the bone marrow rather than in the liver, and that TTV infection was the cause of this patient's aplastic anemia. The precise etiological association of TTV with hepatitis remains to be established.     

深圳地区不同人群TTV感染情况的调查

目的了解深圳地区不同人群ＴＴＶ的感染情况。方法在ＴＴＶＯＲＦ１保守区设计两对套式引物，建立了检测ＴＴＶＤＮＡ的巢式聚合酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ），用该法对深圳地区９０例一般人群、８８例职业献血员、７９例静脉毒瘾者及２９例非甲庚型肝炎病人进行ＴＴＶＤＮＡ的检测。结果ＴＴＶＤＮＡ在以上４种人群中的阳性率分别为７８％，９０％，４１７％与４４８％，前者与后两者比较差异均有显著性（Ｐ＜００５）。９０例一般人群与８８例职业献血员中，１４例ＴＴＶＤＮＡ阳性者丙氨酸转氨酶（ＡＬＴ）均正常。结论深圳地区一般人群与职业献血员中ＴＴＶ携带者较常见；静脉毒瘾者是ＴＴＶ感染的高危人群；部分非甲～庚型肝炎可能与ＴＴＶ相关     

Biliary excretion of TT virus (TTV)

A novel DNA virus (TT virus; TTV) was isolated from a patient with post-transfusion hepatitis of unknown etiology. If TTV replicates in the liver, TTV may appear in the bile. In the present study, to clarify whether fecal-oral infection occur via biliary excretion, the presence of TTV DNA was assessed in paired serum and bile samples collected from 28 patients with obstructive jaundice without parenchymal liver disease. TTV DNA was detected by polymerase chain reaction (PCR) using semi-nested primers, and quantified by Real Time Detection PCR (RTD-PCR). The nucleotide sequence of isolates TTV DNAs was also determined and the sequences were compared between serum and bile samples. Among 28 patients, 7 were positive for TTV DNA in both samples, and 3 and 2 were positive in serum and bile respectively. Of 7 patients positive for TTV DNA in both samples, the TTV DNA titer was higher in serum of 4 patients and in bile of 1 patient. Among 7 patients positive for TTV DNA in serum and bile, 6 had the same sequence in both samples. Multiple distinct types of TTV DNA clones were isolated from serum in 2 patients and from bile in 4 patients. In conclusion, TTV DNA is detected frequently in bile from patients with obstructive jaundice, suggesting a fecal-oral route of infection and high prevalence of asymptomatic TTV carriers. TTV DNA was detected only in serum from some patients, suggesting that replication of TTV may occur in other organs as well as in the liver.     

Detection of TT virus DNA in patients with liver disease and recipients of liver transplant

TT virus (TTV) is transfusion-transmissible but its involvement in post-transfusion hepatitis is uncertain. To investigate the potential association of TTV with liver diseases, the prevalence of TTV DNA was tested by semi-nested PCR in 113 carriers of hepatitis C virus (HCV), 10 patients with acute liver failure, 11 patients with cryptogenic cirrhosis and 200 control blood donors. Thirty-seven of these patients underwent liver transplantation and were tested pre- and post-transplantation. TTV DNA was semi-quantified in serial samples from seven patients with unexplained post-transplant hepatitis. TTV genotyping was performed on samples from 28 patients by sequence analysis. The prevalence of TTV DNA in blood donors was 1.5% and 17% in HCV infected haemophiliacs. In patients with acute or chronic liver disease or hepatitis, 6 to 27% prevalence was observed. After liver transplantation, the prevalence of TTV DNA increased from 16 to 46% (P < 0.01). In patients who developed unexplained hepatitis post-transplantation, TTV viraemia did not parallel ALT levels. TTV DNA either increased in titre or became detectable shortly after transplantation, suggesting that either TTV was transfusion-transmitted, or, more likely, that immunosuppression caused a recurrence of low level or undetectable TTV viraemia. TTV had considerable genomic diversity in the N22 region, corresponding to at least 4 genotypes. Genotype 2 was found in 14/28 patients.     

High detection rates of TTV-like mini virus sequences in sera from Brazilian blood donors

TT virus (TTV) is an unenveloped virus with a single-stranded, circular DNA genome of 3,818-3,853 nucleotides (nt) that infects humans and non-human primates. Recently, the existence of a novel human virus, TTV-like mini virus (TLMV), that shows a genetic organization similar to that of TTV, but with smaller virion particle and genome, was proposed [Takahashi et al. (2000) Archives of Virology 145:979-993]. To date, no information is available with respect to the prevalence and pathogenicity of TLMV. A sensitive PCR assay was developed by using two oligonucleotide primers (LS2 and LA2) designed from the conserved non-coding region of the TLMV genome. One hundred thirty-seven sera from volunteer Brazilian blood donors were tested and 99 (72%) were TLMV DNA positive. No significant differences were observed between the groups of TLMV positive and negative subjects in relation to sex ratio, seroprevalence of TTV DNA, prevalence of anti-hepatitis A virus antibodies, area of residence, occurrence of daily contact with animals, family income, education level, and level of alanine aminotransferase. The specificity of the PCR assay was demonstrated after cloning of amplification products and determination of the nucleotide sequences (200-228 nt) of clones derived from 23 individuals. When DNAs extracted from TLMV/TTV-coinfected sera were submitted to PCR with LS2 and LA2 primers, the amplification products were derived exclusively from the TLMV genome. A markedly wide range of sequence divergence, even higher than that existent among TTV strains, was noted among TLMV isolates, with a maximum evolutionary distance of 0.80.     

Detection rates of TT virus among children who visited a general hospital in Japan

Recently, genomic DNA of the novel TT virus (TTV) was isolated from patients suffering from posttransfusion hepatitis of unknown etiology. We examined sera from 197 children who visited the Department of Pediatrics at Toyohashi National Hospital. Sera were tested for TTV DNA by seminested polymerase chain reaction (PCR) using a set of primers synthesized according to the published TTV sequence. Ten children were found to be positive for TTV (5.1%). All positive PCR products were directly sequenced in both directions using a fluorescent dye terminator cycle sequencing system. The sequences were compared by a multiple sequence alignment and a phylogenetic tree was constructed. The phylogenetic tree showed that two of the TTV isolates found in the present experiment did not belong to any of the phylogenetic groups previously reported.