Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Evaluation of two commercially available nucleic acid hybridization assays for the detection and typing of human papillomavirus in clinical specimens.

Southern blot (Oncor, Gaithersburg, MD) and dot blot (Life Technologies, Gaithersburg, MD) nucleic acid hybridization assays were compared for their ability to detect and type human papillomavirus (HPV) DNA in 50 cervical swab specimens and 11 biopsy specimens. Overall agreement between the two methods was 78.7%. With the use of Southern blot analysis, HPV 6, 11, 16, or 18 was detected in 22 specimens, however, 4 were untypable because of abnormal or smeared band patterns. Dot blot analysis detected HPV 6/11, 16/18, or 31/33/35 in those same 22 specimens and in 9 additional specimens. Eight of the 13 specimens in which HPV was not detected or untypable by Southern blot contained type 31/33/35 by dot blot. Based on convenience of specimen collection and transport, ease of performance and the ability to detect HPV types 31, 33, and 35, the authors are currently using the dot blot assay for the detection and typing of HPV in clinical specimens.     

Atypical cytomegalovirus inclusions in gastrointestinal biopsy specimens from patients with the acquired immunodeficiency syndrome: diagnostic role of in situ nucleic acid hybridization.

Cytomegalovirus (CMV) infection of the gastrointestinal tract is a common cause of morbidity and mortality in the acquired immunodeficiency syndrome. The proper recognition of CMV-infected cells in gastrointestinal mucosal biopsies is critical so that effective therapy is not delayed, preventing further viral dissemination. Although the pathology criteria for classic CMV inclusions have been well described, the occurrence of morphologically atypical inclusions has been reported but the inclusions are not well characterized. This study prospectively examined the relative frequency of classic and atypical CMV inclusions in gastrointestinal mucosal biopsy specimens from 13 human immunodeficiency virus-positive symptomatic patients. The results demonstrated that classic inclusions were rarely found, including four esophageal, one gastric, and one colonic biopsy specimens in which none were seen. However, atypical CMV inclusions were identified from all biopsy specimens examined; these inclusions were much more numerous than classic inclusions and could be categorized into three morphologic types. The atypical inclusions were difficult to precisely identify as CMV-infected cells, but in situ DNA hybridization for CMV was valuable in establishing their viral origin, thus permitting the correct etiologic diagnosis.     

Nucleic acid hybridization for detection of herpes viruses in clinical specimens

A diagnostic hybridization assay for detecting varicella zoster virus (VZV), herpes simplex virus (HSV), and Epstein-Barr virus (EBV) in different clinical specimens was developed using cloned viral DNAs as probes. All probes detected at least 5 pg of homologous DNA and did not cross-react with other viral or cellular DNA. Results of cell culture, serology, and DNA assay were highly concordant. Using a simple standardized protocol for preparation of specimens, hybridization, and washing procedures, this sensitive and specific assay appears to be useful for screening clinical specimens and may be helpful in confirming the serological diagnosis of HSV encephalitis and persistent EBV infections or EBV-associated diseases.     

Rapid enterovirus RNA detection in clinical specimens by using nucleic acid sequence-based amplification

Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.     

Evaluation of NucliSens easyMAG for automated nucleic acid extraction from various clinical specimens

The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n=9) and Chlamydia pneumoniae (n=5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was -2.26 (range, -5.77 to +0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was -3.38 (range, -6.62 to -2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was -0.95 (range, -5.51 to +1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.     

The use of nucleic acid hybridization to detect human coronaviruses

Summary We have applied an RNA: RNA hybridization test for the detection of human coronavirus 229E. This test is undergoing further development but already allows a diagnosis of HCV 229E infection within 48 hours.     

Detection of respiratory syncytial virus antigen and nucleic acid in clinical specimens using synthetic oligonucleotides

Rapid detection of respiratory syncytial (RS) virus in nasopharyngeal secretions (NPS) was carried out on cytospin cell preparations using a directly labelled monoclonal antiserum to RS virus to detect viral antigen and digoxigenin-labelled synthetic oligonucleotides to detect viral nucleic acid. Sequences of 27 and 30 bases in length from within the fusion protein and nucleocapsid genes respectively were selected for use as probes. The oligonucleotide in situ hybridization test was easy to perform and could be completed within 24 hours, but antigen detection was much more rapid and more sensitive. During 1989-1990, more positive results were obtained by antigen detection (193) than by isolation (185), but of 43 confirmed RS-virus-positive specimens, only 21 (49%) were detected by ISH. Antigen detection remains the most suitable single method of rapid detection of RS virus for a diagnostic laboratory.     

Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections

With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.     

Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens

Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBA-beacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.     

Ultrastructural localization of viral nucleic acid by in situ hybridization

In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.     

Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens

The aim of the study was to evaluate the MagNA Pure 96? nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real‐time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96? with 10‐fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty‐nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho‐alveolar fluids, were extracted with the MagNA Pure 96? and the COBAS Ampliprep? instruments. Forty COBAS Ampliprep? positive samples were also tested. Real‐time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96?. Data from clinical respiratory specimens extracted with the MagNA Pure 96? and COBAS Ampliprep? instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1‐2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96? instrument is easy‐to‐use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real‐time PCR assay. J. Med. Virol. 84:906–911, 2012. © 2012 Wiley Periodicals, Inc.     

Improved production of Epstein-Barr virus DNA for nucleic acid hybridization studies.

Large quantities of Epstein-Barr virus (EBV) DNA were prepared by superinfection of Raji cells with EBV. Virus DNA thus prepared showed a single symmetrical peak at 1.718 g/cm³ in CsCl analytical centrifugation and served as a good template for preparation of cRNA specific to EBV DNA. The virus DNA was also highly labeled in vivo with [³H]thymidine or ³²PO₄ for DNA-DNA reassociation studies. The yield of viral DNA from Raji cells, superinfected with EBV prepared from supernatant fluid of HRI cell culture, was 100- to 570-fold higher than that of viral DNA directly extracted from the same virus preparation.     

Dot blot hybridization assay of B19 virus DNA in clinical specimens.

A nonradioactive dot blot hybridization assay for human parvovirus B19 DNA was set up by using a biotin-labeled DNA probe and streptavidin-alkaline phosphatase conjugate. The assay was used to examine 4,895 specimens referred for B19 virus diagnosis during 1987. Of 48 specimens that gave positive reactions for B19 DNA, 41 were confirmed virus positive by electron microscopy (n = 36), radioimmunoassay (n = 26), or counterimmunoelectrophoresis (n = 20). In 7 samples which were not confirmed and in 11 samples giving weak reactions for B19 DNA, there was serological or epidemiological evidence of recent B19 infection. A further 70 specimens gave weak, apparently false-positive reactions. By electron microscopy, 13 of 16 were contaminated by bacteria, and plasmid DNA was demonstrated in one specimen. Of 55 specimens tested, 52 reacted with streptavidin-alkaline phosphatase conjugate alone. These were probable sources of nonspecificity in an otherwise practical and economic screening method for B19 virus.     

Diagnosis of fetal rubella infection by nucleic acid hybridization

The efficacy of nucleic acid hybridization for the diagnosis of rubella infection in experimental and clinical materials was compared with immunoblot and virus isolation techniques. Our results showed that nucleic acid hybridization is specific and rapid but gives false-negative results when compared with conventional virus isolation in some experimental although not in clinical materials so far examined. For this reason, a failure to demonstrate rubella virus in fetal specimens by this method alone cannot yet be taken as a sole criterion for ruling out fetal rubella infection.     

Detection of dengue virus RNA using nucleic acid hybridization

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.     

Direct detection of molluscum contagiosum virus in clinical specimens by dot blot hybridization.

A dot blot hybridization protocol was developed for the direct detection of molluscum contagiosum virus (MCV) DNA in clinical specimens submitted for virus isolation. Samples were concentrated by high-speed centrifugation and treated with proteinase K; this was followed by a single phenol-chloroform extraction step. The DNA was denatured, and the entire volume was spotted onto a nitrocellulose membrane. A biotinylated DNA probe specific for the BamHI-C region of MCV type 1 was used for hybridization. Evidence of MCV DNA was visualized by using streptavidin alkaline phosphatase conjugate and 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as the substrate. Results showed that nonspecific hybridization does not occur with herpes simplex virus- or orf virus-infected clinical specimens and that dot blotting is more sensitive and reproducible than electron microscopy.