沙眼衣原体母婴垂直传播的研究

目的 从基因水平证实沙眼衣原体（ＣＴ）存在母婴间的垂直传播,方法 对１０６对母婴标本（宫颈标本１０６份,婴儿眼结膜,鼻咽部标本２１２份）用培养法证实了ＣＴ的感染,ＰＣＲ扩增了ＣＴ的大部分主要外膜蛋白（ＭＯＭＰ）基因,并做限制性片段长度多态性（ＲＦＬＰ）分析,将母母相对的标本通过ＰＣＲ－ＲＦＬＰ分析法鉴定基因型。结果 １０６例孕妇宫颈标本培养及ＰＣＲ阳性２６例,从２６例阳性孕妇所生新生儿中检出１０例     

母婴垂直传播沙眼衣原体的聚合酶链反应及测序研究

为从分子水平探讨重庆母婴垂直传播沙眼衣原体的发生情况，采用ＭｃＣｏｙ细胞培养及聚合酶链反应技术（ＰＣＲ）检测沙眼衣原体（ＣＴ），并对一母亲宫颈标体及其婴儿鼻咽标本ＰＣＲ扩增出的ＤＮＡ片段克隆后测序。结果：孕妇宫颈ＣＴ培养１０．８％（３０／２７８）阳性，ＰＣＲ阳性率１４．０％（３９／２７８）；母婴垂直传播ＣＴ阳性率５５．０％（１１／２０）；一对母婴标本ＣＴ的ＤＮＡ片段序列完全一致。提示：首次从ＤＮＡ测序分子水平证实母婴垂直传播ＣＴ，垂直传播ＣＴ在重庆地区颇为常见。     

围产期沙眼衣原体感染的研究

为了探讨围产期沙眼衣原体(CT)感染与不良妊娠结局、母婴间的垂直传播及对受染儿的远期影响等问题,我们以直接免疫荧光法、金标法对200例临产妇、25例新生儿(母亲CT抗原阳性),进行了CT抗原检测.结果如下兰州地区产妇中CT的感染率平均为12.5%,母婴间的垂直传播率为40%,产妇感染CT与胎儿早产、出生低体重有相关性.CT在早产儿、低出生体重儿、新生儿窒息者中母婴垂直传播率高于正常足月儿;母婴垂直传播所致受染新生儿今后呼吸系统疾病的感染率高于未受感染儿;直接免疫荧光法、金标法,对CT抗原都是比较准确、快速的检测方法,可以作为早期诊断手段.特别是金标法,更利于在临床中应用、推广围产期沙眼衣原体感染的研究.     

沙眼衣原体母婴垂直感染的研究

探讨沙眼衣原体母婴垂直感染问题。方法;用Ｃｔ单克隆抗体免疫酶检测１８－４９岁１５０名已婚妇女和１８－２６岁１３２名孕妇宫颈分泌物。用ＰＣＲ及Ｃｔ－ＭｃＡｂ－ＨＲＰ两法检测５０例已婚妇女宫颈分泌物,进行阳性与阴性符合率比较。结果;Ｃｔ阳性率已婚妇女为２０．６７％〈孕妇为２０．４５％。     

新生儿沙眼衣原体感染的聚合酶链式反应检测

沙眼衣原体（CT）是一组专性细胞内寄生微生物。是当今世界性传播疾病（STD）中的首位病原体。综合国内各人群组的报道，女性婚检门诊生殖道阳性率为10％，普通妇科门诊为20％，特殊人群（宫颈炎、盆腔炎、性乱者）为50～70％(1)。已构成流行(2)。CT生殖道感染者多数无症状或表现轻微，此类携带者是引起新生儿CT感染的重要来源。据报道，50～70％经有CT感染母亲产道分娩的新生儿可感染。其中20～50％可发生结膜炎，10～20％可发生肺炎，20％粪便可阳性，10～20％女婴阴道拭子阳性。前瞻性研究报迫新生儿CT结膜炎发病率为10～63％活产…     

聚合酶链反应检测新生儿沙眼衣原体肺炎初探

应用聚合酶链反应检测沙眼衣原体ＤＮＡ，８４例新生儿肺炎咽拭子中检出２１例，阳性率２５％，其中剖宫产儿阳性率１６．６％，经产道产儿阳性率２６．４％，早产儿体低重儿阳性率５０％，提示新生儿为ＣＴ感染的高危群体；其感染ＣＴ以通过孕妇产道获得为主。偶有宫内感染；早产低体重儿易被ＣＴ感染且症状较重，ＰＣＲ检测ＣＴ感染具有快速，敏感特异的优点，有助于新生儿ＣＴ肺炎的诊断。     

裂解酶片段长度多态性分析在母婴垂直传播沙眼衣原体主要外膜蛋白基因分型中的应用

目的　建立沙眼衣原体 (Ct)主要外膜蛋白基因 (omp1)上游引物 5′末端地高辛标记裂解酶片段长度多态性 (CFLP)分型方法 ;了解重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型。方法　取临产孕妇宫颈刮片及其新生儿鼻咽拭子组成母婴配对标本 30 0对 6 0 5份 ,采用Ctomp1套式聚合酶链反应 (omp1 nPCR)检测Ct;用建立的CFLP方法对临床Ct分离株进行分型。结果　孕妇宫颈Ct检出率 11% (33/ 30 0 ) ,母婴传播率 2 4 2 % (8/ 33)。生后 2 4h内和 5～ 10d采集Ct阳性母亲所生新生儿鼻咽拭子标本Ct检出率分别为 3 0 % (1/ 33)和 38 9% (7/ 18) ,χc2 =8 79,P <0 0 1。剖宫产和阴道分娩母婴传播率分别为 8 3% (2 / 2 4 )和 6 6 7% (6 / 9) ,χc2 =9 16 ,P <0 0 1。Ct阳性和阴性孕妇胎膜早破发生率分别为 30 3% (10 / 33)和 13 5 % (36 / 2 6 7) ,χ2 =6 4 0 ,P <0 0 5。 8对Ct阳性母婴配对标本CFLP图谱呈 4类 ,经测序证实分别为E、F、H、D型 (各 3、2、2、1对 ) ,各占 37 5 %、2 5 0 %、2 5 0 %和 12 5 % ,且每对母子CFLP图谱完全一致。结论　本研究结果在一定意义上反映了重庆地区近年来临产孕妇Ct感染和母婴垂直传播状况及常见基因型 ;上游引物 5′末端地高辛标记Ctomp1CFLP分型方法灵敏度     

婴幼儿沙眼衣原体肺炎的诊治体会

目的应用PCR法诊断婴幼儿沙眼衣原体(CT)肺炎并探讨不同年龄阶段婴幼儿CT肺炎的临床特点。方法将2007年1月-2009年4月收治的986例0～3岁婴幼儿肺炎作为研究对象,根据年龄分组,对各组婴幼儿均通过咽拭子获取标本,进行CT-DNA测定;总结分析CT-DNA阳性病例。结果986例标本中,CT-DNA阳性64例;其中小婴儿CT肺炎往往无发热,常伴有结膜炎是其特征,而X线多表现为支气管肺炎,其临床表现无特异性。结论CT是婴幼儿肺炎的病原体之一;CT肺炎在3个月内婴幼儿检出率最高;PCR法是检测CT-DNA可靠快捷的方法;大环内酯类抗生素治疗CT肺炎疗效确切。     

沙眼衣原体肺部感染的临床特点

目的　研究沙眼衣原体 (CT)肺部感染的临床特点和诊疗措施。方法　应用聚合酶链反应 (PCR)检测 2 99例住院肺炎患儿呼吸道分泌物中的CT DNA ,其中 62例同时测定血CT IgM。 结果　CT肺炎占同期小儿肺炎的 1 0 .4 % ,1～ 6个月的小婴儿CT肺炎的检出率为 1 4 .8% ,高于 >6个月龄组 6 .7% (χ2 =5 .2 4　P<0 .0 2 5) ;1～ 6个月的无热肺炎中CT肺炎占 2 4 % ,高于有热肺炎组 8.6 % (χ2 =6 .1 1　P <0 .0 2 5)。CT肺炎病程 1 0～ 1 0 1d ,71 %可闻及湿罗音 ,38.7%有哮鸣音 ,45 %白细胞增高 ,X线主要呈支气管肺炎改变。以CT PCR为标准 ,CT IgM阳性一致率 71 .4 % ,阴性一致率 67.3 %。红霉素有效率 85 .7% ,凯福隆为 80 %。结论　CT肺炎多见于小婴儿无热肺炎 ,病程较迁延 ,其他表现缺乏特异性 ,CT IgM诊断CT肺炎特异性不高 ,治疗除红霉素外凯福隆不失为一种选择     

两种方法检测新生儿肺炎沙眼衣原体感染

应用改良McCoy细胞培养法和聚合酶链反应(PCR)检测新生儿肺炎患儿沙眼衣原体(CT)感染。两种方法检测328例新生儿感染性肺炎患儿鼻咽拭子，共证实CT肺炎67例，阳性率为20．4％，以“扩大金标准”来判断结果，PCR的敏感性和特异性分别为98．5％和100％，而培养的敏感性仅89．6％；聚乙二醇(polyethylene glycol，PEG)可提高培养的敏感性。表明CT是新生儿肺炎的重要病原菌。PCR检测CT更敏感、快速。     

新生儿沙眼衣原体肺炎的临床及实验研究

目的探讨新生儿沙眼衣原体（CT）肺炎的发病情况及临床特点。方法对128例新生儿肺炎应用细胞培养检测CT,同时应用PCR扩增其主要外膜蛋白（MOMP）检测CT,应用另一引物对原PCR阳性标本再确证,并分析CT肺炎的新生儿临床资料。结果128例标本中培养阳性29例,阳性率22．7％;两引物PCR均阳性36例,阳性率28．1％;新生儿CT肺炎大多数起病缓慢,临床症状、体征和X线表现无特异性。结论CT是新生儿肺炎常见的病原体,病情迁延,青霉素和头孢类抗生素治疗无效的肺炎应考虑到CT肺炎。细胞培养是诊断CT肺炎的“金标准”,两引物PCR方法的确证可减少因PCR敏感性所致的假阳性。     

Mycobacterium ulcerans infection diagnosed by polymerase chain reaction

Mycobacterium ulcerans infection is the third most important mycobacterial infection world-wide affecting immunocompetent individuals and causes chronic progressive skin ulcers. It has been described in many different regions world-wide. The diagnosis of M. ulcerans infection is often delayed because the diagnosis is difficult to make when new cases appear outside known endemic areas. However, molecular methods are now available to diagnose and distinguish M. ulcerans from other mycobacteria, allowing rapid diagnosis. Presented here is the case of a previously well girl from Townsville, Queensland, with extensive M. ulcerans infection involving the elbow joint, triceps tendon and underlying bone. Rapid diagnosis by polymerase chain reaction confirmed M. ulcerans infection. This is the first known case of M. ulcerans infection from Townsville in over 25 years, highlighting the changing epidemiology of this disease.     

Determination of the six major human herpesviruses in cerebrospinal fluid and blood specimens of children

AIM: To detect and differentiate six major human herpesviruses in the cerebrospinal fluid (CSF) and blood of children by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). METHODS: We synthesized two pairs of primers in the well-conserved regions of the DNA polymerase gene in human herpesviruses. One pair was designed to amplify cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), and the other pair to amplify varicella-zoster virus (VZV) and human herpesvirus 6 (HHV-6) by PCR. Virus species identification was achieved by restriction enzyme digestion with BamHI and BstUI. Ninety-eight CSF and 75 blood specimens were analysed by this technique. At the same time, all blood specimens were also examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirteen (13.3%) of 98 CSF specimens and 26 (34.7%) of 75 blood specimens were positive for herpesvirus DNA in this PCR assay. Only 10 (13.3%) of the blood specimens were positive in ELISA for virus-IgM antibody. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR in detecting herpesvirus infections compared with ELISA were 100% (10/10), 75.4% (49/65), 38.5% (10/26) and 100% (49/49), respectively. These results indicate that the positive rate of PCR was significantly higher than that of ELISA (p < 0.05). The herpesvirus type of these positive specimens was rapidly detected using restriction enzyme digestion with BamHI and BstUI. CONCLUSIONS: PCR-RFLP is a specific, sensitive and accurate technique for the identification of herpesvirus infections in the CSF and blood of children.     

Application of the polymerase chain reaction for the detection of HIV in specimens from newborn screening programs

The use of the polymerase chain reaction (PCR) permits detection of HIV proviral DNA in the universally collected "dried blood spot specimens" of newborn screening programs. Detection of HIV proviral DNA among the annual cohorts of seropositive specimens from ongoing anonymous newborn HIV seroprevalence studies can provide a laboratory-based estimate of maternal-infant transmission rates. Preliminary data suggest that maternal-infant transmission rates may be higher in areas where the newborn seroprevalence rates are highest.     

Molecular Analysis of Hypoxanthine-Guanine Phosphoribosyltransferase Mutations in Five Unrelated Japanese Patients

The isoenzyme of hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) functions in the metabolic salvage of purines. Partial HPRT deficiency is associated with gouty arthritis, while absence of activity results in Lesch-Nyhan (LN) syndrome. We characterized five unrelated patients with HPRT deficiency to understand the spectrum of molecular defects using Southern and Northern blot, polymerase chain amplification of HPRT mRNA and DNA sequencing, and oligonucleotide hybridization analysis of the HPRT gene. Southern blot analysis of DNA indicated that mutations leading to HPRT deficiency in our five patients were not the result of major chromosomal rearrangements or deletions. Sequencing analysis of the amplified DNA from three different patients with HPRT deficiency implied three unique molecular abnormalities: 1) one single-base substitution at codon 54 (from ATG to CTG) resulting in the replacement of methionine with leucine in an LN patient, 2) two single-base substitutions at codon 179 (from GTT to GGT) and at codon 180 (from GGA to AGA) resulting in the replacement of valine with glycine and glycine with arginine in a gouty patient, and 3) 51 nucleotide deletion between nucleotides 747 and 797 resulting in the formation of shorter sized HPRT mRNA and putative two amino-acid deleted HPRT protein in another gouty patient. These results are the direct molecular evidence of genetic heterogeneity in mutant HPRT.     

Prenatal Diagnosis of Duchenne Muscular Dystrophy (DMD) by the Polymerase Chain Reaction (PCR)

The diagnosis of Duchenne muscular dystrophy (DMD) has been drastically improved by recent advances in DNA analysis. The Southern blot hybridization using the cDNA 8 probe and the restriction enzyme Hind III was conducted in a gravida and her family in blood samples. The diagnosis revealed partial gene deletions in both the gravida and the DMD-affected second child. The prenatal diagnosis was performed by studying the PCR (polymerase chain reaction) for target DNAs of exons 48 and 51 that correspond with cDNA 8 probe. In the affected child, the 506 bp band at exon 48 was detected but 388 bp at exon 51 was missing. On the other hand, both the 506 bp band at exon 48 and the 388 bp band at exon 51 were detected in the cultured amniotic cells. Thus, the fetus was determined to be not affected.     

Introductory guide to the language of molecular genetics

BACKGROUND: This special section on molecular genetics was invited in order to raise awareness of the potential of molecular genetic approaches to inform child psychologists, psychiatrists and related professionals. As much of the terminology is specific to the field, this introductory guide aims to aid readers who may be unfamiliar with the approaches of this discipline. METHODS: Basic terminology and genetic processes are described, with sections covering 'The Human Genome' and 'Genetic Markers'. RESULTS: The range of genetic markers is growing all the time, but those that are thought to be functional and/or are highly variable are particularly useful. CONCLUSIONS: Genotyping approaches that offer high efficiency, thus allowing for large numbers of genotypes on large numbers of individuals, are likely to come to the fore in the next few years.     

Polymerase chain reaction for diagnosis of infectious diseases

The polymerase chain reaction (PCR) has been utilized and demonstrated to be useful for detecting minute amounts of a wide variety of infectious agents. In such studies, one must keep in mind that the most appropriate conditions for amplification vary with organisms of interest. In this study, PCR was used as a rapid and sensitive method for detecting infectious agents for which three assay systems were devised comprising the method for the amplification of human T cell leukemia virus type I, Mycobacterium tuberculosis and Mycoplasma pneumoniae. The possibility of vertical transmission of human T cell leukemia virus type I through cord blood was demonstrated using cord blood mononuclear cells from carrier mothers of this virus. In the study of Mycobacterium tuberculosis, PCR was shown to be efficient, particularly in detecting this organism in extrapulmonary cases. Evidence of direct invasion into the central nervous system by Mycoplasma pneumoniae and the concomitant occurrence of mycoplasmaemia in the mycoplasmal central nervous system involvement was obtained using PCR. These results validated the potential of PCR in the clinical research of infectious diseases.