Detection and sequence analysis of the major immediate early and PP150 gene of latent human cytomegalovirus in spleen,liver, and kidney tissues of trauma victims

The presence of human cytomegalovirus (HCMV) DNA in liver, spleen, and kidney samples of HCMV-seropositive trauma victims during latency was demonstrated by polymerase chain reaction (PCR), using primers reactive with the major immediate early gene exon 4 and the structural gene pp150. Sequence analysis of the PCR amplificates showed more than 95% homology with the reference HCMV strain AD169. The few mutations observed were mostly distributed randomly. In one subject two types of the MIE-4 gene were detected, and in another subject two types of the pp150 gene were found, suggesting that different strains of HCMV can be found in organs of the same patient during latency. © 1996 Wiley-Liss, Inc.     

Validation of PCR–reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples

A dermatophyte-specific PCR–reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton , Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.     

傣族、水族和仡佬族人群的α地中海贫血基因突变分析

目的研究傣族、水族和仡佬族人群中常见α地中海贫血基因突变的频率和分布,探讨我国南方少数民族中α地中海贫血的分子流行病学特点。方法AK483例云南省傣族、180例贵州省水族和177例贵州省仡佬族的外周静脉血样本中分离基因组DNA,采用单管多重缺口PCR（Gap-PCR）,进行-α^3.7、-α^4.2和-^SEA三种缺失型α地中海贫血基因突变的检测。结果被检测的傣族、水族和仡佬族样本中α地中海贫血基因突变的携带率分别为34．16％、12．22％和5．08％,以云南傣族的携带率最高。-α^3.7、-α^4.2和--^SHA突变的等位基因频率在傣族中分别是14．18％、0．31％和4．14％,在水族中分别是2．50％、0．28％和3．33％,在仡佬族中分别是1.41％、0．56％和0．56％。-α^3.7是傣族中最常见的突变类型,--^SEA则是水族中的常见突变类型。结论云南傣族、贵州水族和仡佬族中有较高的α地中海贫血基因突变携带率,我国南方不同地区和民族之间在基因突变的频率和分布上均有明显差异。     

Detection of HCV RNA in saliva, urine, seminal fluid, and ascites.

Approximately half of the patients with type C hepatitis do not have a history of parenteral exposure. The route of nonparenteral infection remains unknown. To evaluate the possible role of body fluids, the existence of hepatitis C virus (HCV) RNA in saliva, urine, seminal fluid, and ascites was examined by "nested" polymerase chain reaction (PCR). Amplification of the HCV 5' noncoding sequences was carried out. The amplified product was confirmed by Southern blot hybridization and restriction endonuclease digestion. Among 34 patients with chronic liver disease who were positive for anti-HCV and serum HCV RNA, the prevalence of HCV RNA in body fluids was 100% (7/7) in ascites, 48% (15/31) in saliva, 24% (4/17) in seminal fluid, and 7% (2/29) in urine. The body fluids collected from 3 healthy subjects and 5 patients with chronic liver disease who were positive for anti-HCV but negative for serum HCV RNA were all negative for HCV RNA. Hence, the potential infectivity of body fluids in patients testing negative for serum HCV RNA can probably be discounted. Conversely, the presence of HCV RNA in saliva and seminal fluid of patients positive for serum HCV RNA suggests sexual and household contact as likely modes of nonparenteral transmission of type C hepatitis. Furthermore, the high prevalence of HCV RNA in ascites and saliva may have important implications in medical and dental practice.     

Detection of human herpes virus 8 DNA and sequence polymorphism in classical,epidemic, and iatrogenic Kaposi's sarcoma in South Africa

The aetiology and detection of human herpes virus type 8 (HHV-8) DNA sequences in Kaposi's sarcoma (KS) is a matter of intense investigation. We report on the detection of HHV-8 DNA and sequence polymorphism in different clinicopathological subtypes of cutaneous KS samples from South Africa. The diagnosis was confirmed by histological examination in all cases. Six patients had classic KS (CKS), 3 epidemic KS (EKS), and 3 iatrogenic KS (IKS). A nested polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA in cell lysates, prepared from formalin fixed, paraffin embedded sections. We investigated polymorphism in the HHV-8 DNA using single-stranded conformational polymorphism (SSCP) analysis on the PCR products, followed by direct sequencing. HHV-8 DNA was detected in all the patients with KS, irrespective of the clinicopathological subtype. Direct sequencing was performed on 5 selected cases and showed single base pair substitutions in all. The spectrum of mutations was similar to those described previously. No correlation was found between the different types of KS and sequence variation. The results support the hypothesis that HHV-8 is strongly associated with different clinicopathological subtypes of KS and confirm the occurrence of HHV-8 in patients with CKS, EKS, and IKS in South Africa. J. Med. Virol. 52:168–172, 1997. © 1997 Wiley-Liss, Inc.     

Simultaneous genotyping of single nucleotide polymorphisms in the IL-6, IL-10, TNFalpha and TNFbeta genes

Single nucleotide polymorphisms (SNP) in the human IL-6, IL-10, TNFalpha and TNFbeta genes have been associated with gene function and susceptibility to disease. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a new restriction site recognized by endonucleases AlwNI, BcgI, BglI, BsaBI, BslI, BstXI, EcoNI or XcmI for genotyping SNPs in the IL-6 gene (position - 174), IL-10 gene (positions -592 and -1082), TNFalpha gene (positions -238, - 308 and -863) and TNFbeta gene (position + 249) by mismatched polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Our results show that appropriately designed BslI-based mismatched PCR/RFLP assays can be successfully used to determine the genotypes for approximately 40% of SNPs. The mismatched PCR strategy can be coupled with multiplex-amplification to enable simple and rapid determination of several SNP genotypes in a single reaction.     

Rapid field detection assays for Bacillus anthracis,Brucella spp., Francisella tularensis and Yersinia pestis

Rapid detection is essential for timely initiation of medical post-exposure prophylactic measures in the event of intentional release of biological threat agents. We compared real-time PCR assay performance between the Applied Biosystems 7300/7500 and the RAZOR instruments for specific detection of the causative agents of anthrax, brucellosis, tularemia and plague. Furthermore, an assay detecting Bacillus thuringiensis, a Bacillus anthracis surrogate, was developed for field-training purposes. Assay sensitivities for B. anthracis, Brucella spp., Francisella tularensis and Yersinia pestis were 10-100 fg of target DNA per reaction, and no significant difference in assay performance was observed between the instrument platforms. Specificity testing of the diagnostic panels with both instrument platforms did not reveal any cross-reactivity with other closely related bacteria. The duration of thermocycling with the RAZOR instrument was shorter, i.e. 40 min as compared with 100 min for the Applied Biosystems 7300/7500 instruments. These assays provide rapid tools for the specific detection of four biological threat agents. The detection assays, as well as the training assay for B. thuringiensis powder preparation analysis, may be utilized under field conditions and for field training, respectively.     

Human parvovirus B19 specific IgG, IgA, and IgM antibodies and DNA in serum specimens from persons with erythema infectiosum.

To determine the diagnostic use of different markers of acute parvovirus B19 infection, serum specimens obtained from 128 persons with erythema infectiosum were tested for specific immunoglobulin G (IgG), IgA, and IgM antibodies by capture enzyme immunoassay (EIA) using Chinese hamster ovary (CHO) cell-expressed B19 antigen, and tested for circulating B19 DNA by polymerase chain reaction (PCR). A significant rise in specific IgG and IgA antibodies was detected in 87% and 77%, respectively, of persons from whom acute- and convalescent-phase serum specimens were available. Specific IgA antibodies were detected in single serum specimens from 90% of cases and were present in 22 (18%) of 120 persons from a control group without a history of recent exposure to B19. Specific IgM antibodies were detected in 97% of cases and one person (1%) from the control group. B19 DNA was detected in 94% of cases and was absent in 20 persons from the control group positive for both IgG and IgA antibodies. Serum specimens obtained between 4 and 6 months after onset of illness from six additional persons were also tested. All had specific IgG antibodies, four (67%) had IgA, five (83%) had IgM, and none had detectable B19 DNA. Our data indicate that 1) specific IgA antibodies are too persistent to be a useful indicator of recent B19 infection; 2) specific IgM antibodies are the most sensitive indicator of acute B19 infection in immunologically normal persons but can persist up to 6 months; and 3) B19 DNA can often be detected up to 2 months after onset of illness even in immunologically normal hosts and might be a useful adjunct test for diagnosis of acute B19 infection.     

Human astrovirus, norovirus (GI, GII), and sapovirus infections in Pakistani children with diarrhea

Fecal specimens from 517 infants and young children admitted to the Civil Karachi Hospital, Dow Medical College, Karachi city, Pakistan with acute gastroenteritis from 1990 to 1994 were collected and screened by RT-PCR for human astrovirus (AstV), norovirus (NV), and sapovirus (SV). The specific epidemiological data for illness caused by these viruses in Pakistan are not available. AstV, NV, and SV were detected in 58, 51, and 17 of 517 fecal specimens, and this represented 11.2, 9.9, and 3.2%, respectively. An outbreak of gastroenteritis attributable to AstV serotype 1 was identified during September and October 1990. Moreover, one specimen with a triple mixed infection between AstV (serotypes 1 and 3) and NV GII was found. NV and SV were subjected to molecular analysis by sequencing. One of the sequenced specimens positive for SV turned out to be similar to a strain tentatively called a genogroup IV. The result underscores the importance of these viruses in association with acute gastroenteritis in Karachi city, Pakistan.     

Detection of,and frequent co-infection with,human bocavirus in faecal specimens from children in Wuhan,China

A novel parvovirus, human bocavirus (HBoV), was first discovered in children with respiratory tract infections in Sweden. A retrospective study of HBoV in faecal samples from children suffering from diarrhea, covering a 3-year period (November 2000 to October 2003) in Wuhan, China, was undertaken. PCR assays were used to evaluate 214 faecal samples and to determine the role of HBoV in diarrhoea. Among 196 virus-infected children with diarrhoea, 2.55% were HBoV-positive; however, all HBoV-positive patients were co-infected with common enteric viruses. This result does not support the notion that HBoV is a viral agent causing acute diarrhoea.     

Comparative evaluation of fine needle aspiration cytology, culture, and PCR in diagnosis of tuberculous lymphadenitis

In developing countries, where tuberculosis (TB) is still rampant, tuberculous lymphadenopathy (TB LAP) is one of the most common causes of LAP. Rapid diagnosis and adequate treatment are very important. As a primary diagnostic tool, fine needle aspiration cytology (FNAC) has provided an efficient alternative to excision. Cytologic diagnosis can be made with cytomorphologic features of well-formed epithelioid granulomas and the presence of caseous necrosis. However, bacteriological confirmation is essential because of the presence of various granulomatous inflammation. This study was performed to evaluate and compare the role of FNAC, mycobacterial culture, and PCR in diagnosing tuberculous lymphadenitis. FNA material was collected from 50 patients and was subjected to analysis by cytomorphology, ZN stain, M. tuberculosis culture, and PCR. Out of 50 cases, 36 cases showed cytological features consistent with TB. The most common cytomorphological pattern was epithelioid cell granulomas along with necrosis seen in 17 cases (34%), followed by necrosis only in 13 cases(26%). TBLAP was correctly diagnosed by acid-fast bacilli (AFB) smear in 26 cases, by culture in 30 cases and by PCR in 30 cases. Overall sensitivity of AFB smear was 76.47% and that of culture as well as PCR was 88.23%. In conclusion, presence of granulomas and caseation necrosis are highly suggestive of tubercular etiology, especially in scenario of developing countries where incidence of TB is high. Cytomorphology can be supplemented with AFB smear and culture wherever required and PCR should be kept as a reserve method for equivocal cases.     

VEGF189基因合成、原核表达及鉴定

目的 获得编码VEGF189蛋白的基因,并通过原核表达系统表达VEGF189融合蛋白.方法 应用SOE PCR技术合成VEGF189基因,克隆入pET-32a(+)原核表达载体,转化E coli BL21(DE3)感受态细胞,IPTG诱导表达,进行SDS-PAGE和Western blot检测,表达产物经HisTrapTM HP亲和柱层析纯化,测定蛋白浓度.结果 DNA测序分析表明,合成的VEGF189基因与文献报道相一致.在大肠杆菌BL21(DE3)中表达的Trx-VEGF189融合蛋白,以包涵体和可溶2种形式存在.表达产物可被兔抗鼠VEGF多抗特异识别.经HisTrapTM HP亲和柱纯化,获得纯度达85%的融合蛋白.结论 成功合成VEGF189基因,并在原核表达系统中获得高表达,为研制高效抗肿瘤的VEGF抗体和疫苗奠定了前期基础.     

Multiplex PCR on leptospiral isolates from Kolenchery, Kerala, India

Human leptospirosis causes severe multiorgan dysfunctions that may end in multiorgan failure and death. The methods in hand for diagnosis of leptospirosis like culture, ELISA and MAT only help to confirm the disease, and are of little value in early detection. The aim of this study was to find out if the two sets of primers described earlier could detect all the isolates from the area, for the purpose of using the resultant database for early detection of leptospires in future from clinical specimens. The study was done on culture isolates from Jan 2000 to June 2002 attending the department of medicine, MOSC medical college hospital, Kolenchery, Kerala, India. DNA of 45 culture isolates were amplified by multiplex PCR using two sets of previously described primers, G1, G2 and B 64-I, B 64-II. Specific amplifications of either 285 or 563 bp size were obtained from all isolates included in the study indicating the utility of the multiplex PCR in the rapid detection of leptospires in clinical samples.     

Molecular Diagnostics: Past,present, and future

Molecular diagnostics (MDx) is currently a clinical reality that has its roots deep in the study of gene function, structure, and regulation. The multitude of human mutations identified in the various genetic diseases can now he assayed in the clinical molecular diagnostics laboratory. The polymerase chain reaction (PCR) has facilitated the transition from the research to the clinical laboratory, however, many methods which scan and identify known mutations may not be applicable in a clinical environment. A few of these methods are discussed and one technology that is well suited for clinical use is suggested. Well-trained personnel, regulation of MDx laboratories, and automation are a few of the requirements that will carry us into the promising future of molecular diagnosis. © 1993 Wiley-Liss, Inc.     

Seroprevalence of hepatitis C virus infection and its genotype in Lanzhou,Western China

The seroprevalence of hepatitis C virus (HCV) infection in Lanzhou, Western China was studied. HCV genotypes in 20 patients with HCV infection was determined by genotype-specific primer for polymerase chain reaction (PCR) based on HCV core region and compared with the genotype assigned by sequence comparison and molecular evolutionary analysis based on the same region. Antibody to HCV (anti-HCV) was present in 2.5% of volunteer blood donors and in 35.0% of paid blood donors (P < 0.01). HCV infection is uncommon in patients with liver disease who attended liver clinics in this locality; 4.0% with acute hepatitis and 4.0% with chronic hepatitis, 10.0% with liver cirrhosis, and none with hepatocellular carcinoma were seropositive for anti-HCV. Genotype 1b and 2a were both found to be prevalent. Together, they accounted for 19 of 20 (95%) patients with HCV infection. Sequencing of the HCV core region from two patients showed that the assignment of HCV genotype by genotype-specific primers for PCR matched well with the genotyping results based on sequence comparison and molecular evolutionary analysis. These data showed that HCV is present in Western China, HCV infection is more common in paid blood donors, and HCV genotypes 1b and 2a are both prevalent in Western China. © 1995 Wiley-Liss, Inc.     

SV40-like DNA sequences in pleural mesothelioma,bronchopulmonary carcinoma,and non-malignant pulmonary diseases

Pleural and pulmonary malignancies are usually associated with well-known carcinogen exposure. Recently, the presence of simian virus 40 (SV40)-like DNA sequences has been detected in brain and bone-related human cancers and in pleural mesothelioma. In order to determine whether SV40-like DNA sequences are also present in bronchopulmonary carcinoma and non-malignant lung samples, 125 frozen pleural and pulmonary samples (including 21 mesotheliomas, 63 bronchopulmonary carcinomas, 8 other tumours, and 33 non-malignant samples) and 38 additional samples distant from tumours were studied for the occurrence of SV40-like DNA sequences by polymerase chain reaction (PCR) amplification followed by hybridization with specific probes. Sequences related to SV40 large T antigen (Tag) were present in 28·6 per cent of bronchopulmonary carcinomas, 47·6 per cent of mesotheliomas, and 16·0 per cent of cases with non-neoplastic pleural and pulmonary disease. No statistically significant difference in the occurrence of these DNA sequences was found between malignant mesothelioma and bronchopulmonary carcinoma, but a significantly higher number of mesothelioma cases exhibited SV40-like DNA sequences in comparison with cases of non-malignant pleural or pulmonary disease (P<0·04). Among cases positive for SV40-like DNA sequences, a history of asbestos exposure was found in 3 out of 12 bronchopulmonary carcinomas and 8 out of 10 mesotheliomas. Immunohistochemistry using monoclonal antibodies directed against Tag did not demonstrate nuclear staining. The DNA sequences were not related to BK virus sequences, but three samples were positive with probes hybridizing with JC virus DNA sequences. In conclusion, this study demonstrates the presence of SV40-like DNA sequences in pulmonary neoplasms and in non-malignant lung tissues. It appears that the presence of SV40-like DNA is not unique to cancer. © 1998 John Wiley & Sons, Ltd.     

孕妇血浆中游离胎儿DNA的检测与分析

目的 建立不依赖于胎儿性别和父本DNA、可适用于染色体数目异常和单基因遗传病的无创性产前诊断方法.方法 应用降落PCR和二次PCR扩增技术,联合检测41例正常孕妇(其中孕龄7 w 1例,14～20 w 39例,32 w 1例)血浆中游离胎儿DNA的SRY基因和D17S1293、D21S11、DXS8377等3个短串联重复序列(short tandem repeat,STR)位点.SRY基因扩增产物进行琼脂糖凝胶电泳,STR位点扩增产物进行变性聚丙烯酰胺凝胶电泳并银染显色.结果 41例孕妇血浆DNA中均检出非母源性等位基因条带;联合SRY基因和X-STR位点鉴定胎儿性别,38例判断明确,3例不能明确判断性别.结论 检测孕妇血浆中的游离胎儿DNA,可快捷地获得男性胎儿和女性胎儿的父源性DNA信息,不仅适用于性连锁遗传疾病,而且适用于常染色体遗传疾病等的产前基因诊断.