Detection of Chlamydia trachomatis in culture and urogenital smears by in situ DNA hybridization using a biotinylated DNA probe

The detection of Chlamydia trachomatis by in situ DNA hybridization in urogenital smears was investigated using a commercially available biotinylated DNA probe. Intracellular staining of inclusion bodies was used as the criterion for positivity. Of 35 patients with a culture proven chlamydial infection 19 had smears in which C. trachomatis was detected by in situ DNA hybridization, indicating a sensitivity of 54%. Of 57 patients with a negative culture, two had positive smears by in situ DNA hybridization. To compare whether the criterion for positivity had influenced the sensitivity obtained with in situ DNA hybridization, 14 duplicate smears from culture positive patients were analysed with in situ DNA hybridization and immunofluorescence. Both methods detected intracellular inclusion bodies in seven of these smears, suggesting that the presence of infected cells mainly determines the sensitivity. The DNA probe did not cross-react with micro-organisms commonly found in the urogenital tract.     

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA)

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.     

Detection of herpes simplex virus in clinical specimens by DNA hybridization

An assay to detect herpes simplex virus (HSV) DNA in clinical specimens has been developed. It utilizes nucleic acid hybridization with a 32P-labeled DNA probe prepared from a fragment of HSV DNA cloned in a plasmid vector. This assay can detect 5 X 10(4) plaque-forming units of cell-free HSV and as few as four virus-infected cells. The assay has a sensitivity of 78% and a specificity of 100% compared to virus culture for the detection of HSV in swab specimens from genital lesions. No hybridization is observed with uninfected, varicella-zoster virus infected, or cytomegalovirus infected cells, and specimens from herpes zoster lesions are uniformly negative. While hybridization with a 32P-labeled probe is not optimally suited for routine diagnostic use, this report establishes the feasibility of using nucleic acid hybridization to detect HSV in clinical specimens.     

Detection of varicella-zoster virus DNA in cultured cells and tissue specimens by in situ hybridization using biotin-labeled probes

A method of in situ hybridization with biotinylated probes is described for specific detection of varicella-zoster virus (VZV) DNA in infected cell cultures and in human biopsied or autopsied materials. From molecularly cloned EcoRI-fragments of VZV DNA, we selected as probes for in situ hybridization three fragments (B, H, and K) which had any detectable homology neither with human cell DNA nor with DNAs of VZV-related viruses (herpes simplex virus type 1, type 2 and human cytomegalovirus). The procedure of in situ hybridization was based on that of Brigati et al. (Virology 126, 32-50, 1983), but following modifications were made. 1) The concentration of probe DNA was lowered to 100 ng per ml. 2) Streptavidin-biotin system was used for detection of hybridization. 3) Acid phosphatase was used to generate color development discernible from melanin present in skin. 4) Before hybridization, deparaffinized sections were treated with trypsin by the procedure routinely employed for detection of viral antigens in paraffin-embedded sections.     

Detection of human papillomavirus and human cytomegalovirus in cervical lesions by in situ hybridization using biotinylated probes.

Infections with specific types of human papillomavirus (HPV) have emerged as necessary but not sufficient factors in the development of the majority of cervical cancers. The infection by human cytomegalovirus (HCMV) has also been implicated in both cervical intraepithelial neoplasia (CIN) and cancer. In order to test prevalence of these viral pathogens in genital lesions with suspect cytopathic changes after observation of smears, cervical biopsies from 131 patients were obtained under colposcopic guidance. The biopsies were tested for the presence of HPV and HCMV by the in situ hybridization technique using biotinylated DNA probes on paraffin-embedded sections. Presence of HCMV is twice more frequent in women with HPV-induced cervical lesions (40%) than in women with any detectable HPV (20%). It may be concluded that HCMV might contribute as one synergistic factor in the development of cervical dysplasia.     

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.     

Rapid detection of a specific trimethoprim resistance gene using a biotinylated DNA probe

A DNA probe specific for the dihydrofolate reductase (DHFR) type I gene was labelled with biotin by the process of nick-translation and used to screen 83 independently-derived trimethoprim R plasmids from Enterobacteriaceae. Hybridization was detected using streptavidine and a biotin-conjugated alkaline phosphatase to generate an insoluble coloured precipitate following the addition of an appropriate dye. Sixty-eight plasmids (81.9%) hybridized with the probe for DHFR type I. The method could be adapted for use with any antibiotic resistance gene for which a suitable DNA probe is available and has none of the drawbacks associated with the use of radioactively-labelled DNA in hybridization techniques.     

脆弱拟杆菌DNA探针在临床上的应用

目的：在DNA水平上准确鉴定B.fragilis(脆弱拟杆菌）。方法：用脆弱拟杆菌种特异的探针pBF-15与分离株或标本进行DNA点杂交。结果：8株生化鉴定为脆弱拟杆菌的7株经DNA探针证实为脆弱拟杆菌;1株粪拟杆菌、1株吉氏拟杆菌和2株未能鉴定到种的拟杆菌经探针重新鉴定的为脆弱拟杆菌。用pBF-15直接检测了46例临床标本,其结果与常规法符合。结论：DNA探针鉴定脆弱拟杆菌或诊断脆弱拟杆菌感染比常规法准确、快速、简便。     

Effects of post-mortem autolysis on the detection of rabies virus genomic RNA and mRNA in mouse brain by using in situ hybridization.

The effects of post-mortem autolysis were studied on the detection of rabies virus RNA in the brains of mice with experimental rabies by using in situ hybridization (ISH). The brains of CVS-infected mice were subjected to autolytic periods in situ of up to 72 h. ISH was performed with 3H-labelled RNA probes for rabies virus glycoprotein gene genomic RNA and mRNA. During the post-mortem period there was progressive loss of signals for genomic RNA and mRNA, which was greater for mRNA. ISH signals in perikarya also changed for genomic RNA from a multifocal to a diffuse distribution during the post-mortem period. Rabies virus antigen was better preserved during the autolytic period. Effects of the agonal state, degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues.     

DNA probe versus culture for detection of Mycoplasma pneumoniae in clinical specimens

The laboratory diagnosis of Mycoplasma pneumoniae is often difficult because of lengthy and complicated cultural methods and serological tests that may be both insensitive and nonspecific. In this study, 82 patients suspected of Mycoplasma pneumonia were cultured for M. pneumoniae, and their respiratory secretions were tested by a DNA probe for M. pneumoniae. The probe test was 100% sensitive and 98% specific compared to culture. This DNA probe, then, is an effective alternative method for the detection of M. pneumoniae in respiratory specimens.     

TTV感染的肝组织病理学特点及病毒DNA原位检测

目的 观察单一ＴＴＶ（ｔｒａｎｓｆｕｓｉｏｎ ｔｒｓｎｓｍｉｔｔｅｄ ｖｉｒｕｓ）急性感染患者肝组织病理损害特点及病毒ＤＮＡ的表达。方法 对某职业学校ＴＴＶ感染流行期间,１８例血清ＴＴＶ阳性住院患者的肝活检组织进行ＨＥ、浸银Ｇｏｍｏｄｒｉ染色检查,以及地高辛标记ＴＴＶ ＤＮＡ（Ｄｉｇ－ＴＴＶ ＤＮＡ）探针原位杂交。结果 １８例肝穿刺标本中出现汇管区炎１４例（７７．８％）,胆小管损害６例（３３．４％）,肝小叶内灶状坏死７例（３８．９％）,微脂滴３例（１６．７％）,汇管区纤维化２例（１１．１％）,原位杂交阳性８例（４４．４％）,ＴＴＶ阳性表达细胞散在分布于肝小叶内,坏死区及汇管区旁较密集。结论 单一ＴＴＶ急性感染可致患者肝组织轻度炎性病变,其特征是汇管区炎及胆小管损害。ＴＴＶ可能是一种嗜肝病毒。     

The detection of Mycobacterium tuberculosis in uncultured clinical specimens using the polymerase chain reaction and a non-radioactive DNA probe.

A Sal I-Hin dIII restriction fragment from Mycobacterium tuberculosis was found to hybridize specifically with genomic DNA from M. tuberculosis. Primers were designed from the sequence of this fragment and used to amplify uniquely M. tuberculosis-group DNA in a polymerase chain reaction. It is suggested that a combination of these primers and an acetylaminofluorene-labelled probe will prove to be a useful tool for the early diagnosis of tuberculous infections.     

Diagnosis of hydatidiform moles by polymorphic deletion probe fluorescence in situ hybridization

Because products of conception often contain maternal and villous tissues, the determination of maternal and villous genotypes based on genetic polymorphisms can help discern maternal and paternal chromosomal contribution and aid in the diagnosis of hydatidiform moles. Polymorphic deletion probe (PDP) fluorescence in situ hybridization (FISH) probes based on copy number variants are highly polymorphic and allow in situ determination of genetic identity. By using three informative PDPs on chromosomes 2p, 4q, and 8p, we compared maternal with villous genotypes and determined the ploidy of villous tissue. PDP FISH was performed on 13 complete moles, 13 partial moles, 13 nonmolar abortions, and an equivocal hydropic abortion. PDP FISH permitted definitive diagnosis of complete moles in five of 13 cases for which maternal and villous genotypes were mutually exclusive. A complete mole was highly suspected when all three PDP loci showed homozygous villous genotypes. The diagnosis of a complete mole by PDP FISH yielded a theoretical test sensitivity of 87.5%, specificity of 91.8%, an observed test sensitivity of 100%, and specificity of 92.3%. Triploidy was observed in all partial moles, in which diandric triploidy was confirmed in six cases. In the equivocal hydropic abortion, PDP FISH combined with p57 immunofluorescence revealed placental androgenetic/biparental mosaicism. PDP FISH can be used in clinical practice and research studies to subclassify hydatidiform moles and evaluate unusual products of conception.     

Detection of rabies virus mRNA in mouse brain by using in situ hybridization with digoxigenin-labelled RNA probes.

A non-isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues. Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used. The method had good sensitivity and low backgrounds, and there was excellent cellular localization of signals. ISH wih digoxigenin-labelled probes was compared with ISH with 3H-labelled probes. This non-isotopic method of ISH is more convenient than the radiolabelled method, and it is quicker because a long autoradiographic exposure is not required.     

Rapid detection of herpes simplex virus DNA by in situ hybridization with photobiotin-labelled double-stranded DNA probes

An assay for rapid detection of herpes simplex virus in infected cells is described. The assay utilizes in situ hybridization with photobiotin-labelled double-stranded DNA probes prepared from HSV-1 DNA cloned in plasmid vectors. The assay provided an alternative method for earlier detection of virus in cell cultures with the ease of preparation of photobiotin-labelled double-stranded DNA.     

Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum

A 5.5 kilobase DNA fragment from an Eco RI digest of the Mycoplasma gallisepticum genome was specific for the detection of M. gallisepticum. This 5.5 kb fragment was initially cloned into bacteriophage lambda gt11 followed by subcloning into the plasmid vector pGEM-3Z. The incorporation of a biotin label was accomplished by utilizing biotin-11-dUTP in a nick translation reaction. This probe, designated pMg6, reacts specifically with M. gallisepticum when tested against various mycoplasma DNAs in Southern blot hybridization analysis. Spot-blot hybridization data indicate the pMg6 is capable of detecting 800 pg of M. gallisepticum DNA.     

Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples

Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.     

荧光原位杂交检测宫颈上皮脱落细胞人端粒酶基因表达与人乳头瘤病毒的临床应用

目的研究宫颈癌人端粒酶基因(TERC)的表达情况和宫颈癌人乳头瘤病毒(HPV)感染的相关性,评估荧光原位杂交(FISH)技术检测TERC基因的表达对子宫颈上皮内瘤样病变(CIN)发展到宫颈癌的预测价值。方法采用FISH技术进行子宫颈上皮脱落细胞TERC基因的检测,实验对象81例,病理正常组20例,CIN1组28例,CIN2组12例,CIN3组9例,宫颈癌组12例。同时利用实时荧光定量聚合酶链反应技术(FQPCR)检测这81例病人的HPV(16/18)感染情况。并进行宫颈癌与TERC基因和HPV的相关性分析。结果在不同病理分组中TERC基因检测和HPV检测的阳性率差异有统计学意义(P<0.05),TERC基因检测的阳性率在不同病理分组中的差异有统计学意义(P<0.05),HPV的阳性率在不同病理分组中的差异也有统计学意义(P<0.05),恶性程度越高差异越显著(P<0.01);宫颈癌组TERC基因异常表达阳性率是100%,HPV为91.7%。结论 TERC基因异常表达与宫颈癌的发生发展及HPV感染密切相关。TERC基因和HPV联合检测,有助于提高宫颈癌的早期诊断有重要价值。