In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts

1. The identification of cytokine genes expressed in the central nervous system is critical to understanding the immune network in various diseases of brain, such as infection, degeneration, and malignancy. 2. Expression of cytokine genes in human astrocytoma cell lines and in fresh brain specimens was studied by the reverse-transcribed/polymerase chain reaction method. 3. The correlation between clinical malignancy and cytokine gene expression within malignant glioma was examined, especially regarding the relevancy of inhibitory cytokines, such as transforming growth factor-beta and interleukin-10.     

Characterization of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type-specific cDNA probes

Dot and Northern blot hybridization assays were used to analyze field strains of group A bovine rotaviruses (BRVs) by using nucleic acid probes representing P and G type specificities. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions of the cloned VP4 (nucleotides 211 to 686) and VP7 (nucleotides 51 to 392) genes from four serotypically distinct (in P or G types) strains of rotaviruses: NCDV (G6, P1), IND (G6, P5), 69M (G8, P10), and Cr (G10, P11). The P and G type cDNA probes were radiolabeled with [32P]dCTP and hybridized with RNA extracted from reference cell culture-passaged rotavirus strains or the field samples. The field samples were obtained from young diarrheic calves from Ohio, Nebraska, Washington State, and Canada. The cDNA probes were specific for their respective G or P types on the basis of analysis of known P and G type reference strains. The G typing analysis of 102 field samples revealed that 36.3% (37 of 102) were G6, 2.9% (3 of 102) were G8, 12.7% (13 of 102) were G10, and 23.5% (24 of 102) were untypeable. The P typing results for 93 samples indicated that 2.2% (2 of 93) were P1 (NCDV-like), 20.4% (19 of 93) were P5 (UK-like), 9.3% (10 of 93) were P11 (B223-like), and 40.8% (38 of 93) were untypeable. This is the first report of the identification among BRV strains in North America of a G type other than G6 or G10. Our report further confirms that G6, P5 rotaviruses are predominant among the BRV field strains that we examined, and the P types of these strains differ from that of the BRV vaccine strain used in the United States (G6, P1). The large number of untypeable G (23.5%) and P (40.8%) types suggests that other or new P and G types exist among BRV field strains.     

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes

F-specific (F+) RNA coliphages are prevalent in sewage and other fecal wastes of humans and animals. There are four antigenically distinct serogroups of F+ RNA coliphages, and those predominating in humans (groups II and III) differ from those predominating in animals (groups I and IV). Hence, it may be possible to distinguish between human and animal wastes by serotyping F+ RNA coliphage isolates. Because serotyping is laborious and requires scarce antiserum reagents, we investigated genotyping using synthetic oligonucleotide probes as an alternative approach to distinguishing the four groups of F+ RNA coliphages. Oligoprobes I, II, III, IV, A, and B were selected to detect group I, II, III, IV, I plus II, and III plus IV phages, respectively. Methods for phage transfer from zones of lysis on a host cell lawn to candidate membrane filters and fixation of genomic nucleic acid on the membranes were optimized. The oligoprobes, which were end labeled with digoxigenin, were applied in DNA-RNA hybridization, and hybrids were observed by colorimetric, immunoenzymatic detection. Of 203 isolates of F+ RNA coliphages from environmental samples of water, wastes, and shellfish, 99.5 and 96.6% could be classified into each group by serotyping and genotyping, respectively. Probes A and B correctly identified 100% of the isolates. On the basis of these results, this method for genotyping F+ RNA coliphages appears to be practical and reliable for typing isolates in field samples.     

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease

PURPOSE: The common finding of thrombi between the bifoil balloons when they were extracted after mitral dilation prompted us to look for evidence of minor brain embolisms using the sensitive technique of BMRI (brain magnetic resonance T2-weighted imaging). METHODS: BMRI was performed within 48 hr before and after a percutaneous mitral balloon commissurotomy (PMBC) in each of the 63 patients in this study. RESULTS: There was evidence (hyperintensity foci: HI) of a previous asymptomatic brain embolism in 38 of 63 patients before PMBC and a new HI appeared in 18 of 63 patients after the procedure. New HI signals were found exclusively in the white matter in 8 of 18 patients and in only 3 of 18 were HI signs larger than 1 cm. One patient, with an HI signal >1 cm in the thalamus and another <1 cm in the brain stem, presented diplopia accompanied by other minor clinical signs. The differences in HI rate among four subgroups (1, older vs younger than 43 years; 2, sinus rhythm vs atrial fibrillation; 3, echo score <8 vs >8; 4, patients from western countries vs the others) were not statistically significant, probably because the number of patients in each subgroup was low. Patients in atrial fibrillation had slightly more (not significant) HI before PMBC (15/20, 75%) than patients in sinus rhythm (23/43, 53%), but after PMBC their HI frequencies were similar (atrial fibrillation: 5/20, 25%; sinus rhythm: 13/43, 30%). CONCLUSION: Brain microembolism is frequent during PMBC, but is often anatomically limited and free from clinical signs in most cases. Brain embolism seems to be related mainly to the procedure itself and not the features of the patient.     

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.     

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.     

RNA extraction from virus-diseased sweet potato for reverse transcriptase polymerase chain reaction analysis

Apoptosis occurs in both clinical and experimental alcoholic liver disease. The mechanisms involved in alcohol-induced apoptosis of liver cells are not completely understood. Induction of cytochrome P450 2E1, the alcohol-inducible cytochrome P450, is one of the proposed mechanisms. Exposure of Hep G2 cells expressing cytochrome P450 2E1 to arachidonic acid leads to increased lipid peroxidation and apoptosis. Increased levels of iron in the liver also promote lipid peroxidation and are associated with increased numbers of apoptotic hepatocytes. Tumor necrosis factor (TNF) acting through its receptors can induce apoptosis in hepatocytes. Increased levels of tumor necrosis factor and its receptors have been described in alcoholic liver disease. The liver is also CD95 receptor positive and in liver tissue from patients with alcoholic hepatitis, the CD95 ligand is expressed at high levels in hepatocytes. Cytotoxic T lymphocytes could, through the CD95 receptor-ligand interaction, promote apoptosis.     

Demonstration of Epstein-Barr viral DNA in paraffin-embedded tissues of Burkitt's lymphoma from Argentina using the polymerase chain reaction and in situ hybridization

Burkitt's lymphoma (BL) is the most frequent non-Hodgkin's lymphoma in children in Argentina. Although epidemiologic studies have linked Epstein-Barr virus (EBV) to more than 90% of African BL cases but to only 10-20% of American and French cases, increased EBV-specific antibody titers were demonstrated in 73% of Argentinian patients with BL. To characterize the relationship between EBV and BL in Argentina, we analyzed paraffin-embedded tissues from 16 cases of BL for the presence of EBV DNA using the polymerase chain reaction (PCR) and in situ hybridization (ISH). PCR analysis showed that only 4 of 16 specimens contained the EBV BamW fragment, and these specimens were all from cases diagnosed in 1984. Results of ISH performed with a specific biotinylated DNA probe against the NotI/PstI fragment of EBV correlated with the PCR findings. EBV sequences were detected with ISH in 70-90% of the tumor cells from the 4 positive cases. These data may suggest an epidemic outbreak of EBV-related BL in 1984 superimposed on sporadic cases of BL, for which EBV may not have been an essential factor. This study also demonstrates the value of using molecular techniques on archival tissue to track epidemiologic trends.     

Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.     

Detection of hepatitis C virus with RNA polymerase chain reaction in fulminant hepatic failure

The role of hepatitis C virus (HCV) infection in fulminant hepatic failure is controversial. The frequency of serum HCV RNA positivity in previously reported patients with fulminant hepatic failure (FHF) of indeterminate cause ranged from 0 to 12% in the United States and Europe and from 43% to 59% in Asia. We assessed serum HCV RNA using polymerase chain reaction (PCR) and oligoprimers from the 5'UTR of the HCV genome in 26 consecutive patients with FHF. Another laboratory independently performed PCR on 21 of the serum samples using different oligoprimers from the 5'UTR and NS3 region of the HCV genome. Serum HCV RNA was detected in two of seven (28%) patients with hepatitis B, 9 of 15 (60%) with an indeterminate cause, and in none with hepatitis A (n = 2) or drug-induced hepatotoxicity (n = 2). HCV RNA PCR results were concordant between both laboratories in 17 of 21 (81%) of samples. In patients with an indeterminate cause, HCV RNA positivity was significantly associated with the transmission risk factor of low socioeconomic status and Hispanic ethnicity. Eighteen patients underwent liver transplantation (LT) and 15 (83%) survived. Among patients with FHF of indeterminate cause, recurrent or acquired HCV infection after transplantation occurred in three of five (60%) and one of four (25%) patients, respectively. Three of four (75%) patients with hepatitis C virus infection post-LT also developed histologic hepatitis. HCV appears to be the causative agent of a substantial number of cases of FHF classified as indeterminate in the Los Angeles area. Differences in patient populations or risk factors may explain the discordant incidences of HCV infection in FHF observed among different programs.     

Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction

We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.     

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.     

Rapid isolation of cell-type-specific protein tyrosine kinases by degenerate polymerase chain reaction combined with differential hybridization technique

To identify protein tyrosine kinase (PTK) genes preferentially expressed in renal cell carcinoma cell line, we screened a PTK-cDNA-enriched library constructed from RNA of an renal cell carcinoma cell line with a PTK probe, each produced from renal cell carcinoma, gastric cancer or esophageal cancer cell lines by degenerate polymerase chain reaction. Two cDNA fragments of PTK genes, FRK and FLT-3, were isolated from the PTK-cDNA-enriched library of the renal cell carcinoma cell line by differential hybridization technique. The FRK cDNA clone represented 15.8% of the PTK-cDNA-enriched library from the renal cell carcinoma cell line, while the FLT-3 cDNA clone was 2.8% of the same library. Both of the two PTK genes were expressed preferentially in renal cell carcinoma cell lines. This method, described here, is useful for the rapid isolation of PTK cDNA fragments, including a low abundant cDNA, preferentially expressed in a specific cell line.     

Specific amplification of Aspergillus fumigatus DNA by polymerase chain reaction

Herpes simplex (HSV) and varicella-zoster (VZV) skin infections share so many histological similarities that distinguishing between them may prove to be impossible. We developed and characterized a new monoclonal antibody, VL8, IgG kappa isotype, directed to the VZV envelope glycoprotein gpI. Immunohistochemistry with VL8 appeared highly sensitive and specific on formalin-fixed paraffin-embedded biopsies and a clear-cut distinction between HSV and VZV infections was possible. The pattern of VL8 immunolabelling in VZV infections was strikingly different from that found in HSV infections studied with polyclonal antibodies to HSV I and II. Double immunolabelling revealed the VL8 positivity of sebaceous cells, endothelial cells, Mac 387- and CD68-positive monocyte-macrophages, and factor XIIIa-positive perivascular, perineural and interstitial dendrocytes. Intracytoplasmic VL8 labelling of endothelial cells and perivascular dendrocytes was found at the site of leukocytoclastic vasculitis.     

Detection of human papillomavirus in routinely processed biopsy specimens from laryngeal papillomas: evaluation of reproducibility of polymerase chain reaction and DNA in situ hybridization procedures

The frequency of human papillomavirus (HPV) in laryngeal papillomas varies largely among different studies. DNA in situ hybridization (ISH) has been the most widely used method for detection of HPV. The aim of this study was to compare the reproducibility and sensitivity of ISH with polymerase chain reaction (PCR) in 35 specimens of laryngeal papillomas routinely fixed in buffered or unbuffered formalin. Out of 12 specimens fixed in buffered formalin, 10 were positive for HPV 6/11 using ISH. The procedure was repeated three times and three specimens were positive only twice. Nine biopsies were positive for HPV using PCR with consensus primers (My 09/11) on dewaxed tissue without extracting DNA. In three repeated PCRs, the results were inconsistent in three samples. After DNA extraction, all 12 samples were positive with PCR. Of the 23 specimens fixed in unbuffered formalin, 14 were HPV-positive with ISH, while only one was positive with PCR. We concluded that PCR with My 09/11 consensus primers is a highly sensitive method for detection of HPV in laryngeal papillomas fixed in buffered formalin, but useless for samples fixed in unbuffered formalin. When DNA was extracted from the former type of fixed tissue, the results were highly reproducible. In contrast to PCR, ISH appeared to be less influenced by fixation procedure, but it was not as reproducible and sensitive as PCR. Negative results did not necessarily mean absence of HPV.