特异性引物聚合酶链反应乙型肝炎病毒基因分型法的建立及应用

目的应用型特异性引物聚合酶链反应法（PCR）进行乙型肝炎病毒基因分型并分析该法的可靠性。方法应用型特异性引物PCR和INNO-LiPA分别对深圳、长春、北京152份HBV DNA阳性慢性乙型肝炎患者的血清标本进行了基因型分型，对该两种分型法不一致的血清标本再进行S区基因测序分型，以确定该两法的可靠性。结果型特异性引物PCR和INNO-LiPA的总符合率为86．8％（132／152），不一致率为13．2％（20／152）。型特异性引物PCR检测到81份（53．3％）B型；58份（38．2％）C型；13份（8．5％）B＋C型混合感染，未检出其他基因型或混合感染的基因型。INNO-LiPA检测到74份（48．7％）B型；61份（40．1％）C型；5份（3．3％）B＋C型混合感染；另检出3份（2．0％）A＋B型混合感染；1份（0．7％）B＋E型混合感染；1份（0．7％）C型与D型，1份（0．7％）D型感染，3份（2．0％）B／C／D型及3份未能分型。20份两法分型不一致的标本中，6份无剩余血清，对其余14份进行了S区基因测序分型，结果型特异性引物PCR与S区基因测序分型法的符合率为71．4％（10／14），而INNO-LiPA与S区测序法的符合率仅为7．1％（1／14），前者明显高于后者（P〈0．05）。结论型特异性引物PCR和INNO-LiPA均可鉴定HBV基因型，但前者较为简便和可靠，且费用较低，可用于临床标本的检测和流行病学调查。     

北京地区人乳头瘤病毒16型感染及其E6/E7基因变异与宫颈病变的相关性研究

目的探讨HPV16感染及其E6/E7基因变异与宫颈病变的相关性。方法采用导流杂交技术进行HPV感染分型检测,PCR扩增出80份HPV16阳性宫颈病变的E6/E7基因、克隆入pMD18-T载体,双向测序分析基因变异与宫颈病变相关性。结果HPV16在宫颈病变患者中的检出率最高为33.3%(154/463),与病变程度相关(P<0.05)。E6/E7基因72份测序成功,DNA序列变异发生率为88.9%(64/72)。氨基酸序列E6-D32E(T96G)和E7-N29S(A86G)位点突变同时伴随存在,D32E/N29S的检出率为38.9%(28/72),与宫颈病变程度相关(P<0.05)。结论HPV16是北京地区来源的宫颈病变中最常见的致病型,其D32E/N29S变异与病变程度相关。     

mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。     

卡氏肺孢子虫的分类、命名、体外培养及其动物模型的建立

以往所称的“卡氏肺孢子虫” (Pneumocystiscarinii,Pc)能感染包括人和实验动物在内的多种哺乳动物 ,免疫力低下的宿主感染后能引起致命的卡氏肺孢子虫肺炎 (Pneumocystiscariniipneumonia,PCP )或称肺孢子虫病(Pneumocystosis)。国际上已将原感染人体的卡氏肺孢子虫 (Pneumocystiscarinii)更名为Pneumocystisjeroveci,国内张瑞娟、朱淮民( 2 0 0 3)将其译为耶氏肺孢子虫 ,然尚未被广泛应用。其生物学分类也由原来动物界的原虫定为真菌界的真菌类。重新命名和跨界的分类归属具有重要意义 ,然而 ,国内有关“卡氏肺孢子虫”的报道 ,尚未启…     

贵州侗族、苗族和汉族人群乙型肝炎病毒基因型分布

目的　了解贵州侗、苗、汉族HBV感染者的基因型。方法　比较 12 7株各基因型HBV全序列S基因核苷酸序列 ,用DNA分析软件筛选出 3个限制性内切酶。设计 3条HBVS区引物并进行聚合酶链反应扩增 ,产物经MboⅠ、BstNⅠ或BsmAⅠ酶切 ,分析酶切产物电泳图谱 ,建立区分HBV(A～F)基因型的方法。对贵州 16 6份侗、苗、汉族HBV感染者血清进行基因分型 ,5份PCR产物直接测序验证分型方法的准确、可靠。结果　S基因PCR RFLP分型结果准确 ,5份酶切鉴定结果经测序证实。 16 6份标本中 ,B基因型 138份 (83 13% ) ,C型 2 8份 (16 87% ) ,未发现B、C以外的其他基因型。侗族的 4 8例中 ,4 7例为B型 (97 92 % ) ,1例C型 (2 0 8% ) ,苗族的 5 2例中 ,4 9例为B型(94 2 3% ) ,3例C型 (5 77% ) ;6 6例汉族也以B基因为主 (6 3 6 4 % ,4 2 6 6 ) ,但C型有 2 4例 (36 36 % ) ,与侗、苗族HBV感染者相比 ,差异有显著意义 (χ2 =35 0 5 88,P <0 0 0 5 )。结论　贵州地区HBV基因型由B、C 2型构成 ,且以B基因型为主。汉族患者C型较多 ,而侗、苗族患者中B型是占绝对优势的基因型。     

五条蚋和双齿蚋nrDNA-ITS区序列分析(双翅目:蚋科)

测定我国两个常见吸血蚋种五条蚋和双齿蚋各 4个克隆nrDNAITS区 (包括ITS1、ITS2和 5 8SrRNA基因 )序列及其两侧的 18S和 2 8SrRNA基因部分序列。两蚋种 5 8SrRNA基因大小均为 12 2bp。五条蚋ITS1和ITS2大小分别为 136bp和 32 3bp。双齿蚋ITS1和ITS2大小分别为 10 1～ 10 5bp和 32 1～ 32 4bp。五条蚋不同克隆ITS1 ITS2拼接序列的同源性为 99% ,双齿蚋则为 96 %～ 10 0 % ,显示五条蚋和双齿蚋nrDNA存在重复变异型     

拉米夫定联合阿德福韦酯治疗慢性乙型肝炎应答不佳者基因型演变特点研究

目的 了解拉米夫定初始联合阿德福韦酯治疗慢性乙型肝炎应答不佳者基因型特点及其演变规律.方法 应用克隆测序法检测3例患者(S1患者、S2患者、S3患者)拉米夫定初始联合阿德福韦酯治疗12个月以上病毒学应答不佳慢性乙型肝炎患者基线、治疗4周、12周、24周、48周、60周HBV基因型,每个时间点各随机挑取25个克隆进行鉴定并测序.结果 3例初始联合拉米夫定和阿德福韦酯治疗慢型乙型肝炎应答不佳患者各时间点的总克隆数为398份,其中S1患者在基线时C基因型(8.3％)和B基因型(91.7％)共同表达,但B基因型占绝对优势(22/24).治疗60周时C基因型占绝对优势(100％).S2和S3患者在基线时仅表达B基因型,在治疗过程中,B基因型逐渐“漂移”为C基因型,治疗60周时,C基因型占绝对优势(S2中占75％,S3中占100％).结论 克隆测序可以更好的反应整体的基因型水平；在长期药物压力下HBV基因型从B基因型逐渐向C基因型演变,是导致拉米夫定初始联合阿德福韦酯治疗应答不佳的主要原因.     

福建省新分离3株乙型脑炎病毒的分子特征鉴定

目的　确定福建省新分离乙型脑炎病毒 (JEV)的基因分型及其E区段氨基酸序列特征。方法　用逆转录聚合酶链反应 (RT PCR)扩增并克隆新分离JEV(0 2 4 1、0 2 4 3、0 2 10 2 )的PrM、E区段核苷酸序列 ,测序后应用ClustalX软件做碱基配对和比较分析 ,种系发生采用PHYLIP软件包分析。结果　新分离的 3株JEV属于基因Ⅲ型 ,E区段核苷酸和氨基酸与减毒活疫苗株SA 14 14 2株的同源性均在 96 %以上 ,在关键结构域有部分氨基酸差异。结论　福建省新分离的 3株病毒属于基因Ⅲ型JEV ,E蛋白与疫苗株相比有部分氨基酸差异     

河北某非法采血村人群感染HCV基因亚型调查

目的 了解河北某非法采血村村民感染丙型肝炎病毒(HCV)基因亚型分布情况.方法 对该村全体村民采集静脉血标本计520份,无菌分离血清;以RT-PCR法扩增其中可利用的483份血清HCV之C/E1基因片段,双脱氧链终止法测序;用Mega4.0软件进行HCV系统进化分析,构建系统进化树,判定基因亚型.结果 483份标本中,HCV RNA阳性者70份,阳性率14.5%;基因分型1b亚型36例,占51.4%;2a亚型34例,占48.6%.结论 该村村民血清HCV检出率约为14.5%,远高于一般人群;HCV亚型构成为1b、2a各半.     

广州地区小儿无症状乙肝病毒携带者的基因型研究

目的扩增HBV DNA S基因,建立HBV DNA的分型方法,研究广州地区小儿无症状乙肝病毒携带者(AsC)的基因型. 方法利用PCR-RFLP基因分型方法,扩增HBV DNA S基因,以限制性内切酶AvaII、MboI酶切分型. 结果 60份AsC血清样本,B型37份(62.7%),C型20份(33.3%),B、C混合型2份(3.3%),未分型1份(1.7%),经序列分析证明为C型,C型共35%. 结论 PCR-RELP分型方法经济实用,分型率达98.3%,广州地区小儿乙肝病毒的基因型以B型和C型为主,其中携带者中B型占优势.     

Genetic diversity of Pneumocystis carinii f. sp. hominis based on variations in nucleotide sequences of internal transcribed spacers of rRNA genes

A variety of genes have been used to type Pneumocystis carinii. In the present study, nucleotide sequence variations in the ITS1 and ITS2 internal transcribed spacer (ITS) regions of the rRNA genes were used to type Pneumocystis carinii f. sp. hominis DNA obtained from the lungs of 60 human immunodeficiency virus-infected individuals. These regions were amplified by PCR, cloned, and sequenced. Multibase polymorphisms were identified among samples. Several new genotypes are reported on the basis of the nucleotide sequence variations at previously unreported positions of both the ITS1 and the ITS2 regions. Twelve new ITS1 sequences were observed, in addition to the nine sequence types reported previously. The most common was type E, which was observed in 60.5% of the samples. The sequence variations in the ITS1 region were mainly located at positions 5, 12, 23, 24, 45, 53, and 54. Sixteen new ITS2 types were also identified, in addition to the 13 types reported previously. The most common was type g (26.6%). The sequences of the ITS2 regions in most specimens were different from the previously published sequence at bases 120 and 166 through 183. The most common variations observed were deletions at positions 177 through 183. The presence of more than one sequence type in some patients (60%) suggested the occurrence of coinfection with multiple P. carinii strains. The genetic polymorphism observed demonstrates the degree of diversity of Pneumocystis strains that infect humans. Furthermore, the high degree of polymorphism suggests that these genes are evolving faster than other genes. Consequently, the sequence information derived is useful for purposes such as examination of the potential of person-to-person transmission and recurrent infections but perhaps not for other genotyping applications that rely on more stable genetic loci.     

Genotyping and coalescent phylogenetic analysis of Pneumocystis jiroveci from South Africa

Sequence analysis of Pneumocystis jiroveci internal transcribed spacer (ITS) regions has become an important epidemiological tool. The objectives of the present study were to investigate sequence variations in the ITS1-5.8S ribosomal DNA (rDNA)-ITS2 regions; determine the P. jiroveci genotypes present in Cape Town, South Africa; and resolve the lineage evolution of the types by use of the coalescent theory. ITS regions were amplified from samples collected from 19 patients. PCR products were cloned, and four to five clones were sequenced from each specimen. Statistical parsimony was applied for coalescence-based network genotype analysis. The most prevalent type was Eg (14 of 19 patients, 33 of 83 clones), followed by Gg (4 of 19 patients, 7 of 83 clones), Eu (3 of 19 patients, 5 of 83 clones), and Gh (2 of 19 patients, 2 of 83 clones). Four new combinations (Eo, Je, Ge, and No), 11 new ITS1 sequences, and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and was designated type u. Coinfection appeared to be common, with 15 of 19 patients harboring more than one type and with up to six types per specimen. The resultant parsimony network identified Eg as the most probable ancestral haplotype and supported the occurrence of recombinational events within the population studied. Although the 5.8S rDNA region revealed only 13 clones containing one to two nucleotide polymorphisms, it may assist in defining types. Coalescent theory proposed that Eg is an ancestral type from which microevolutionary subtypes radiate.     

Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States

Fecal specimens were obtained from 12–24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.     

Typing of Pneumocystis carinii strains with type-specific oligonucleotide probes derived from nucleotide sequences of internal transcribed spacers of rRNA genes.

We have recently developed a method for typing Pneumocystis carinii strains that infect humans. The method takes advantage of nucleotide sequence variations in internal transcribed spacers (ITSs) of the rRNA genes of P. carinii. To date, two types of nucleotide sequences (designated types A and B) have been found in the ITS1 region, and three types of nucleotide sequences (designated types a, b and c) have been found in the ITS2 region. Of the six potential combination types, we have detected four, designated types Ac, Bb, Ba, and Bc. To simplify typing, we have designed five oligonucleotide probes, probes 1-A, 1-B, 2-a, 2-b, and 2-c, which are specific to ITS1 type A and type B and ITS2 type a, type b, and type c, respectively, of P. carinii strains that infect humans. We also have designed an oligonucleotide which reacts specifically with P. carinii strains that infect rats. The ITS region were amplified by PCR, and the PCR products were then probed with these type-specific oligonucleotide probes. Typing with the type-specific oligonucleotide probes was found to be effective with specimens containing only one type of P. carinii. These methods are rapid and simple to perform and will be useful for studying the epidemiology of P. carinii infections.     

Detection and genotyping of Epstein-Barr virus by polymerase chain reaction in tissues obtained from cases with Hodgkin's disease in Turkey.

In order to determine the positivity rate and genotype of Epstein-Barr virus (EBV) in cases with Hodgkin's disease (HD) in Turkey, 40 tissue specimens from HD patients were analysed. Ten non-lymphoid tissue samples from individuals without any evidence for lymphoma were used as controls. The cases with HD included 33 males and 7 females with a mean age of 28 years. Nodular sclerosis was the most prevalent histological subtype (16/40) followed by mixed cellularity (10/40), lymphocyte predominance (9/40), and lymphocyte depletion (5/40). After histopathological evaluation, deparafinisation and lysis of the specimens, one-stage polymerase chain reaction (PCR) and two-stage (nested) PCR assays were performed with the primers common for both EBV genotypes and the primers specific for EBV types 1 and 2, respectively. EBV DNA was detected in 22 of 40 (55%) cases with HD and in 1 of 10 (10%) control specimens. The distribution of EBV DNA positivity according to the histological subtypes was as follows: 10 of 16 (62.5%) for nodular sclerosis, 3 of 5 (60%) for lymphocyte depletion, 5 of 9 (55.6%) for lymphocyte predominance, and 4 of 10 (40%) for mixed cellularity. Although most of the HD patients were males of 15-34 years of age, there were no significant differences between EBV positivities obtained from different sex and age groups. The rates of EBV genotypes were 82% for type 1, 9% for type 2, and 9% for both types, respectively.     

Genetic distribution of group A human rotavirus types isolated in Gyunggi province of Korea, 1999-2002.

BACKGROUND: Human rotavirus genotypes G1-G4 and G9 are the major etiological agents of infantile gastroenteritis. G1 was the most prevalent in Korea during the 10-year period prior to 1997. However, between 1998 and 1999, G4 was the predominant type in Korea, as it was in other Asian countries. OBJECTIVES: The circulating pattern and genetic variability of group A human rotavirus in Gyunggi, Korea, 1999-2002, were examined in 189 stool specimens. STUDY DESIGN: Stool samples were collected from children with diarrhea, and group A human rotavirus type was determined using multiplex RT-PCR in those specimens found to be positive for rotavirus by ELISA. Each genotype was sequenced, and phylogenetic analysis was performed on the sequences. RESULT: We found significant variability from year to year in the prevalence of different G and P types of rotavirus. We also found relatively high prevalence rates for types normally considered to be uncommon. Furthermore, we found that the most prevalent combination of G and P types changed from year to year. Although the combination of G and P types changed every year, the sequence of G genotypes showed a high level of similarity (>97%) compared to those of strains from other Asian countries. CONCLUSION: We report the types of rotavirus circulating in Gyunggi province, Korea from 1999 to 2002. This information on rotavirus diversity has important implications for rotavirus vaccine efficacy and future vaccine development.     

Internal transcribed spacer regions of rRNA genes of Pneumocystis carinii from monkeys

Analysis of sequence variations among isolates of Pneumocystis carinii f. sp. macacae from 14 Indian rhesus monkeys (Macaca mulatta) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from P. carinii f. sp. hominis, the ITS sequences from various P. carinii f. sp. macacae isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike P. carinii f. sp. hominis infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of P. carinii f. sp. macacae. These results offer insights into the genetic differences between P. carinii organisms which infect distinct species.     

Occurrence and genotypic characteristics of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in pigs with diarrhea

There is limited information available on the association between Enterocytozoon bieneusi and diseases in animals or on the characteristics of the strains involved. This study examined the occurrence of E. bieneusi in piglets with and without diarrhea to determine its involvement. Among 472 fecal samples from 472 piglets (237 with diarrhea and 235 without) up to 7 weeks of age, 67 (approximately 14%) were polymerase chain reaction (PCR) positive for E. bieneusi. Of the 237 piglets with diarrhea, 38 (approximately 16%) tested positive for E. bieneusi. Of the 235 healthy piglets, 29 (approximately 12%) tested positive for E. bieneusi. This species was detected only in the younger group of piglets with diarrhea, particularly those aged less than 1 week and between 1 and 2 weeks. This suggests that E. bieneusi is a possible cause of diarrhea in piglets. This organism, however, produced asymptomatic infections in the older piglets, as there was no significant difference in the rates of occurrence between the diarrheic and nondiarrheic older piglets (aged older than 4 weeks). The internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid gene of the ten E. bieneusi-positive samples was amplified using nested PCR and subsequently sequenced. Genetic polymorphisms, which were represented by five distinct genotypes (PEbA–PEbE), were found among the E. bieneusi isolates. The five genotypes identified in this study differed from each other by two to six single-nucleotide polymorphisms. Nine isolates from four genotypes (PEbA–PEbD) were homologous to previously known types that had originally been isolated from pigs. However, one isolate from the PEbE genotype was identical to type CAF1, which was originally isolated from humans. In addition, the phylogenetic relationships determined by the neighbor-joining analysis of the ITS sequences indicated this genotype to be more distant from the other pig-specific genotypes. Thus, this isolate from pigs may be distantly related to the pig-specific genotypes and may be capable of infecting humans.     

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.     

Genotyping of Chlamydia trachomatis strains from cultured isolates and nucleic acid amplification test-positive specimens

Urogenital strains of Chlamydia trachomatis are divided into several serogroups (D-K). Since these serovars are represented with differing prevalence in the population a serotyping of strains is necessary, when characterising the epidemiological situation. The aim of this study was the genotyping of C. trachomatis strains, the comparison of the results with those of serotyping, and the genotyping of positive specimens using commercial nucleic acid amplification tests (NAAT). The Chlamydia trachomatis major outer membrane protein gene (omp1) from 55 isolated strains and 36 NAAT-positive specimens was amplified by polymerase chain reaction (PCR). The restriction fragment length polymorphism (RFLP) patterns of these amplicons were compared with those of reference strains. The genotypes E and F were found to be most prevalent. The results are discussed considering other studies, genovariants and epidemiology.