Spot Hybridization Technique Research Articles

A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction

A rapid single-step procedure for the isolation of low molecular weight DNA using guanidinium thiocyanate and phenol as protein denaturants is described and applied for the detection of specific hepatitis B virus (HBV) DNA sequences from serum samples by the polymerase chain reaction (PCR). The novel technique is efficient and, when compared to the standard proteinase K/phenol/chloroform method has the advantage of being faster and easily adaptable to the routine processing of a high number of clinical samples by PCR and spot hybridization techniques.

Serum hepatitis B virus DNA detects cryptic hepatitis B virus infections in multitransfused hemophilic patients

The recognition of replicating hepatitis B virus (HBV) may be important to both define the cause of and know how to manage chronic liver disease in multitransfused hemophilic patients. Replicating HBV can be detected at the molecular level by methods for HBV-specific DNA (HBV- DNA), which are much more sensitive than the immunologic methods for detecting hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Unselected hemophilic patients (260; 6% with HBsAg, 4% with isolated anti-hepatitis B core (anti-HBc), 52% with anti-HBs and anti- HBc, 26% with isolated anti-HBs, and 12% with no HBV marker) were investigated retrospectively with a dot spot hybridization technique that detects serum HBV-DNA down to 0.5 pg and by Southern blot analysis, which tests the specificity of the HBV-DNA reactions. Eighteen patients (7%; five with serum HBsAg and 13 HBsAg seronegative with antibodies to HBV) had serum HBV-DNA. Serum HBV-DNA was detected more frequently in HBsAg carriers than in seronegative patients (33% versus 6%, P less than .01), and had no relationship to serum alanine aminotransferase. Serum HBV-DNA was more sensitive than the radioimmunoassay for HBeAg was for detecting replicating HBV (7% versus 1.1%, P less than .01). These findings demonstrate that there is cryptic HBV infection in a number of hemophiliacs and that serum HBV- DNA may coexist with markers thought to reflect immunity against HBV.

Serum Hepatitis B Virus DNA Detects Cryptic Hepatitis B Virus Infections in Multitransfused Hemophilic Patients

The recognition of replicating hepatitis B virus (HBV) may be important to both define the cause of and know how to manage chronic liver disease in multitransfused hemophilic patients. Replicating HBV can be detected at the molecular level by methods for HBV-specific DNA (HBV-DNA), which are much more sensitive than the immunologic methods for detecting hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Unselected hemophilic patients (260; 6% with HBsAg, 4% with isolated anti-hepatitis B core (anti-HBc), 52% with anti-HBs and anti-HBc, 26% with isolated anti-HBs, and 12% with no HBV marker) were investigated retrospectively with a dot spot hybridization technique that detects serum HBV-DNA down to 0.5 pg and by Southern blot analysis, which tests the specificity of the HBV-DNA reactions. Eighteen patients (7%; five with serum HBsAg and 13 HBsAg seronegative with antibodies to HBV) had serum HBV-DNA. Serum HBV-DNA was detected more frequently in HBsAg carriers than in seronegative patients (33% versus 6%, P less than .01), and had no relationship to serum alanine aminotransferase. Serum HBV-DNA was more sensitive than the radioimmunoassay for HBeAg was for detecting replicating HBV (7% versus 1.1%, P less than .01). These findings demonstrate that there is cryptic HBV infection in a number of hemophiliacs and that serum HBV-DNA may coexist with markers thought to reflect immunity against HBV.

Detection of hepatitis B virus DNA in serum by spot hybridization technique: Sensitivity and specificity of radiolabeled and biotin-labeled probes

The detection of serum hepatitis B virus (HBV) DNA in 32 chronic HBsAg carriers was performed by spot hybridization technique using both biotinylated and radiolabeled probes, in order to compare their specificity and sensitivity. Our results show that both assays are specific since neither evidence of cross-hybridization between HBV DNA and sera from patients with chronic non-A, non-B (NANB) hepatitis was found, nor HBV DNA was detected both in patients with chronic anti-HBs/anti-HBc-positive hepatitis and in patients negative for all HBV markers. An agreement between the two assays was observed in 94% of the tested sera. Even though in 5 serum samples (6%) low levels of HBV DNA (0.1-1 pg/100 microliter) remained undetected using the biotin-labeled probe, the lower detection limit of the two assays (0.1 pg of HBV DNA) was the same using purified Dane particles as control. This study indicates that the enzymatic detection of HBV DNA is suitable for routine and rapid monitoring of HBV replication in both HBeAg- and anti-HBe-positive patients, as well as for a semiquantitative analysis of serum HBV DNA.

Detection of Hepatitis Delta Virus in Serum and Liver Tissue by Molecular Hybridization: Validation of a Rapid Spot-Hybridization Technique

Serologic tests for detection of delta hepatitis virus (HDV) antigen and antibody have recently been supplemented with a Northern blot hybridization assay for HDV RNA. However, this technique is cumbersome for analysis of multiple samples. In order to simplify detection of HDV RNA, the authors have tested a spot-hybridization method with a new HDV cDNA probe. Their method has proved to be rapid, sensitive, and specific for HDV RNA even when less ultracentrifugation was used for recovering serum RNA. Results for HDV RNA were concordant by both spot and Northern blot hybridization in 12 serum samples from patients with known delta hepatitis, whereas in seven cases spot-hybridization was superior in detecting liver HDV RNA. The concordance between HDV RNA by spot hybridization and delta antigen was complete, whereas that between delta IgM (44% overall) or IgG (67% overall) was less strong. The authors' observations indicate that this new technology permits detection of HDV RNA with relative ease and could be applicable to the evaluation of large numbers of cases with delta hepatitis.

Nucleic acid spot hybridization with nonradioactive labeled probes in screening for human papillomavirus DNA sequences.

We examined 161 human tissue samples using the spot hybridization technique with nonradioactive labeled DNA probes of human papillomavirus (HPV). Whole cells were spotted on nitrocellulose filters; DNA of the cells was denatured and fixed to the filter. Then the DNA spots were hybridized to nonradioactive labeled DNA and monitored by a sandwich immunoenzymatic reaction. This technique is simple, sensitive, specific, requires no special equipment, and can be used in clinical settings. HPV DNA was found in 92% of samples in which, on the basis of histologic and colposcopic criteria, its presence was suspected, as well as in 31 samples where it was not suspected.

Relationship between hepatitis B virus deoxyribonucleic acid, HBeAg/anti-HBe status in serum and HBcAg in liver: its clinical significance in chronic HBsAg carriers.

Sera and biopsied liver specimens from 45 hepatitis B surface antigen (HBsAg) carriers both with or without overt liver diseases and negative for anti-delta antibody, were examined for markers of hepatitis B virus (HBV) infection: hepatitis B e antigen (HBeAg), hepatitis B virus deoxyribonucleic acid (HBV-DNA) in serum and hepatitis B core antigen (HBcAg) in liver. HBV-DNA in serum was assayed by the spot hybridization technique, and HBcAg in the liver was investigated by the peroxidase anti-peroxidase method. Among the parameters showing active replication of HBV, serum HBeAg, serum HBV-DNA and intrahepatic HBcAg were found in 27 (60%), 27 (60%) and in 22 (48.9%) of 45 HBsAg carriers, respectively. The presence of serum HBV-DNA and of intrahepatic HBcAg in HBeAg positive cases and the absence of serum HBV-DNA and of intrahepatic HBcAg in anti-HBe positive cases was the rule with few disparate cases among those with chronic liver disease. These parameters were not useful in predicting the histology on liver biopsy. The activity of hepatic inflammation in the HBsAg chronic carriers assessed by serum alanine aminotransferase level closely paralleled HBV-DNA in serum but not HBeAg in serum, HBcAg in liver or histologic picture on biopsy. HBV-DNA may be the most sensitive parameter of replication of hepatitis B virus and of the activity of inflammation in the liver.

Transmission of Three Viroids Through Seed and Pollen of Tomato Plants

AbstractThe severe strain of potato spindle tuber viroid (s‐PSTV) as well as chrysanthemum stunt (CSV) and cucumber pale fruit (CPFV) viroids were found to be transmitted through seed and pollen of the tomato cvs. Rutgers and Najwcześniejszy. Plants pollinated with a pollen infected with any of these three viroids became systematically infected. Plant, fruit and seed symptoms of viroid infection were noted on sap‐ and pollen‐inoculated plants and the yield of these plants was reduced.Tomato cv. Rutgers plants grown from infected seeds were symptomless although all three viroids were detected in these plants by bioassay and by electrophoresis on 5% polyacrylamide gel.When DNA complementary to s‐PSTV RNA was used for a direct viroid detection in seed samples by spot hybridization technique it hybridized not only with s‐PSTV RNA but also with CSV RNA as well as with CPFV RNA.

Histopathological analysis of chronic hepatitis B virus (HBV) infection in relation to HBV replication.

The interrelationship among expression patterns of hepatitis B surface and core antigens (HBsAg and HBcAg) in the liver, hepatitis B virus (HBV) DNA in sera, HBeAg/anti-HBe status and histological features was examined in 189 liver specimens and 106 sera from Japanese patients with chronic HBV infection, utilizing immunoperoxidase methods and a spot hybridization technique. HBsAg and HBcAg were distributed uniformly among the lobules in 8 viral carriers with "normal" liver (NVC) and 30 patients with persistent hepatitis (PB) seropositive for HBcAg. This uniform staining pattern was quite distinct from the non-uniform and irregular patterns in 137 patients with chronic active hepatitis (CAH) associated with or not associated with cirrhosis. HBcAg was often found to be stained strongly in the cytoplasm as well as in the nucleus of hepatocytes in HBeAg-positive CAH, in contrast to NVC/PH, in which cytoplasmic HBcAg was very weak. Serum HBV DNA was detected in all 22 cases with HBeAg-positive NVC/PH, 41 of 46 (89.1%) cases with HBeAg-positive CAH, 6 of 23 (26.1%) casew with anti-HBe-positive CAH and none of 3 cases with anti-HBe-positive NVC/PH. The level of serum HBV DNA and staining of HBcAg were in decreasing order in these groups. While the presence of serum HBV DNA and HBcAg staining was always associated with HBeAg seropositivity in NVC/PH, this was not always found in CAH. Moreover, necroinflammatory activity in the liver did not always parallel viral replication in CAH. These findings seem to confirm that NVC/PH and CAH have different biologic processes; viral replication is always concordant in the former, but is sometimes discordant in the latter with HBeAg/anti-HBe status. The immunologic response of the host seems to suppress and distort replication of the virus to a varying degree among patients and areas of the same liver in CAH differently to that in NVC/PH. The different life cycle of the virus, including integration of viral DNA into the cellular genome, may subsequently result in discordant states of various virus markers in CAH.

Cryptic hepatitis B virus replication during prednisone therapy in type B chronic active hepatitis.

Hepatitis B virus (HBV) replicating in patients with serum hepatitis B surface antigen and antibody to hepatitis B e antigen (cryptic HBV replication) can be detected by a spot hybridization technique for serum HBV-DNA and by immunoperoxidase staining of hepatitis B core antigen in the liver. These methods allowed us to study the effects of chronic (13 to 82, mean 30 months) administration of low doses (10 or 15 mg/day) of prednisone to 17 patients with chronic active hepatitis and cryptic HBV replication. Liver biopsies performed before treatment demonstrated that 1 to 50% (mean 12%) of the liver cells were infected. After therapy, infected cells had disappeared in 5 (29%), were considerably reduced in 9 (53%) and remained unchanged in 3 (18%) patients. The mean percentage of infected cells in the liver biopsies performed at the end of the follow-up was 3.2 +/- 5.5% (p less than 0.005). Serum HBV-DNA was present in 12 of 13 and in 5 of 12 patients investigated before treatment and at the end of the study, respectively. Five patients harboring HBV in the liver developed cirrhosis during treatment. Our data indicate that, despite steroid therapy, HBV replication either ceased or was decreased in two thirds of the patients, while in no case it flared-up. The rise of cirrhosis was not prevented by this type of therapy.

Quantitation of hepatitis B virus dna (HBV DNA) in serum using the spot hybridization technique and scintillation counting

The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 μl) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a ‘Hybri.Dot’ system. Quantification of HBV DNA by 32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the ‘Hybri.dot’ was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0–800 pg HBV DNA) than optical densitometry measurements (0–50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948; P <0.001) but the assay proved more sensitive since HBV DNA (> 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116–303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.

Discordance of hepatitis B e antigen/antibody and hepatitis B virus deoxyribonucleic acid in serum: Analysis of 1063 specimens

Hepatitis B virus DNA was determined in 1063 serum samples from 252 patients with hepatitis B surface antigen by the spot hybridization technique. The results were correlated with hepatitis B e antigen and antibody. Hepatitis B virus DNA was detected in 87% of hepatitis B e antigen-positive patients, in 18% of hepatitis B e antibody-positive patients, and in 18% of those negative for both. Discordance of antigen/antibody and hepatitis B virus DNA, i.e., the presence of the DNA in antibody-positive sera or the absence of the DNA in antigen-positive sera, was observed in 209 of 997 (21%) samples. Of 121 patients with histologic diagnosis, this discordance was observed in none of 20 patients with nonspecific changes, in 13% of 39 with chronic persistent hepatitis, in 21% of 38 with chronic active hepatitis, and in 38% of 24 with cirrhosis. Thus, hepatitis B e antigen/antibody testing alone failed to predict the presence or absence of circulating hepatitis B virion in a significant proportion of patients with advanced chronic liver disease.

Analysis of DNA polymerase reaction products for detecting hepatitis B virus in serum—comparison with spot hybridization technique

An assay for DNA polymerase reaction products using slab gel electrophoresis and autoradiography was compared with the spot hybridization technique for the detection of hepatitis B virus DNA in 317 blood samples. The former could identify the nature and size of DNA on electrophoresis, and reduce potentially false-positive results due to artifacts. Discordant results between the two methods occurred in 36 of 317 samples; 22 were positive by the spot technique alone, and 14 were positive by the analysis of DNA polymerase reaction products alone. However, the samples positive with the spot test alone showed weak radioactive signals on electrophoresis/autoradiography that were often interpreted as "inconclusive" by blind observations. Correlation of hepatitis B e antigen/antibody with hepatitis B virus DNA was studied in 91 patients with various chronic liver diseases. Discordant results, i.e., presence of the DNA in antibody positive sera, or its absence in the antigen positive sera, were obtained in 15 (19%) cases. Such patients tended to have advanced liver disease with fluctuating serum aminotransferase levels. Analysis of DNA polymerase reaction products by slab gel electrophoresis and autoradiography is not only sensitive, but is also as specific as the Southern blot technique in the detection of hepatitis B virus DNA in serum, and may prove useful in selected samples, especially where no cloned hepatitis B virus DNA is available, or in search of new hepatitis B virus-like viruses.

Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum.

Hepatitis B virus DNA (HBV DNA) in serum was measured by a Spot hybridization technique in a consecutive series of 79 cases with chronic HBV infection from Taiwan. HBV DNA was found in 96.3% (52/54) of HBeAg-positive, 66% (2/3) with neither HBeAg or anti-HBe and in 63.6% (14/22) of anti-HBe positive patients. The levels of HBV DNA in the HBe-Ag-positive patients were significantly higher than in the anti-HBe positive patients (median, 944 vs. 58 pg per ml, p less than 0.001). The mean ages increased from 28.7 years for the cases with high levels of HBV DNA, to 34.7 years for those with low levels (p less than 0.01) and to 41.0 years in those without HBV DNA in serum (p less than 0.05 when compared with those with low level of HBV DNA). Ninety per cent of patients (27/30) with high levels of HBV DNA showed only minor hepatic inflammatory activity, as did 91% (10/11) of those without HBV DNA. In contrast, histologic signs of chronic active hepatitis or chronic lobular hepatitis were demonstrated in 76% of cases (29/38) with low levels of HBV DNA. These data are consistent with the hypothesis that liver damage occurs during the period of clearance of hepatocytes supporting HBV replication, and are inconsistent with the view that HBV may be directly cytopathic. Thus, the natural history of chronic HBV infection may be divided into three phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid diagnosis of plant virus infections by spot hybridization

Spot hybridization techniques can be used to identify plant viruses in sap extracts. This new and rapid diagnostic technique offers greater sensitivity and better discrimination between similar viruses than is available through existing serological assays. Furthermore spot hybridization is much more rapid, and requires much less labour, than current biological tests.

Hepatitis B virus DNA (HBV-DNA) in anti-HBe positive sera.

HBV-DNA measured by the spot hybridization technique, was found in the sera of 28 of 106 (26.4%) anti-HBe positive carriers of HBsAg. Dane particle-associated HBeAg, HBcAg and HBV-specific DNA-polymerase activity were found in the sera of nine (8.5%), five (4.7%) and two (1.9%) of these patients, respectively. All carriers with serum HBV-DNA had chronic liver disease and 18 had intrahepatic delta-Ag and serum anti-delta at titers higher than 1/5000. Intrahepatic HBcAg was detected in the nuclei of 90% of delta negative individuals; 50% of them also had cytoplasmic fluorescence. Only two of the 18 patients with intrahepatic delta-Ag (11%) had HBcAg in the liver. Viral nucleic acid was not found in the sera of 15 other patients with chronic hepatitis, seven of whom had intrahepatic delta-Ag. Serum HBV-DNA was also negative in the remaining 63 symptomless carriers of HBsAg lacking markers of delta infection. Interestingly, although DNA-polymerase negative, some sera gave autoradiographic spots of high optical density. HBV-DNA was detected in them at concentrations typical of sera which are usually both DNA-polymerase and HBeAg positive. Detection of HBV-DNA in serum represents the most direct and sensitive in vitro assay for assessing HBV infectivity and characterizes HBsAg carriers with HBV-related liver damage and ongoing HBV replication independently from the state of HBeAg/anti-HBe system. In the Mediterranean area, the majority of anti-HBe positive carriers with serum HBV-DNA have chronic liver disease and delta infection.

Spot hybridization: Application to viroid identification

Summary A cloned 32 P-labelled cDNA probe to potato spindle tuber viroid (PSTV) was used to investigate the possibility of applying the spot hybridization technique to the identification of 2 virois PSTV and chrysanthemum stunt viroid (CSV). The best conditions for obtaining the highest sensitivity consisted of spotting heat-denatured samples onto the nitro-cellulose membrane, then hybridizing at 42 or 55°C in the presence of 50% formamide. The technique led to detection of 100–250 pg of purified viroid RNA and to identification of the viroid in a plant extract corresponding to 0.1 mg of infected leaves. These values demonstrated a clear advantage of the hybridization technique over the electrophoretic assay. A 3 H-labelled probe could be substituted for the 32 P probe, but it exhibited poor sensitivity due to the initially low specific radioactivity of the 3 H probe. Despite large sequence homologies, the PSTV 32 P-labelled probe hybridized only with CSV in purified solutions under non-stringent conditions, e. g. low temperature (42°C) and in the presence of relatively high amounts of CSV (50 mg). Therefore, spot hybridization appeared to be a more sensitive diagnostic technique than the electrophoretic assay, but with a high degree of specificity even with apparently closely related viroids.

Detection of Hepatitis B Virus DNA in Serum by a Simple Spot Hybridization Technique: Comparison with Results for Other Viral Markers

A simplified spot method for determination in serum of hepatitis B virus DNA (HBV DNA) by molecular hybridization is proposed. For simultaneous testing of 30 serum samples, it reduced to about 1 hr the duration of the steps preceding hybridization proper. The method also greatly reduced the loss of DNA during these steps and allowed more sensitive detection in samples of only 25 or 50 microliters. HBV DNA was determined in 181 serum samples by this method, and the results were pooled with 67 previous determinations by the Southern blot technique. Results for the pool were then compared to those obtained with radioimmunoassay for serological HBV markers. Ninety-six of the 248 samples were HBV DNA positive. Eleven others gave variable or inconclusive results, probably due to low viral particle titers. Seventy-two HBsAg- and HBeAg-positive sera contained HBV DNA, confirming that HBeAg is a marker of active viral replication. Fourteen other HBsAg- and HBeAg-positive sera, obtained from eight patients, were either HBV DNA negative or oscillated between negative and positive, or, again, were weakly positive; serological follow-up in 7 patients showed seroconversion to anti-HBe in 5, 3 of which became HBsAg negative. Eight of the HBsAg-positive sera were negative or borderline for HBeAg but contained HBV DNA and may, therefore, have been infective; seven of these sera had anti-HBe. Six HBsAg-negative sera contained HBV DNA and may also have been infective; five of these exhibited HBV antibodies. These results indicate that molecular hybridization not only provides a more sensitive and direct method for detecting hepatitis B virus in serum but also defines additional serological patterns with predictive or epidemiological value.

Hepatitis B virus replication and clinical outcome in carriers of HBsAg. Perspectives of treatment with DNA inhibitors.

HBV-DNA was measured by the spot hybridization technique in serial serum samples obtained from 47 HBsAg carriers followed up for a mean of 4 years. The levels of HBV-DNA were compared to the conventional HBV serology and immunopathology to determine the relation of active HBV replication to the outcome of hepatitis and the suitability of Italian HBsAg carriers for treatment with DNA inhibitors. HBV-DNA was found in 26 carriers (53%) and persisted with comparable serum levels in 24 of them throughout the follow up. The occurrence rate of an unfavorable outcome as determined by histological evidence of cirrhosis was 6% versus 44% (p less than 0.01) in carriers with active viral infection (greater than 1 ng/ml of HBV-DNA) and in patients with absent or low levels of viral DNA (less than 1 pg/ml), respectively. Progression of the liver disease could not be predicted on the basis of active HBV replication and was presumably related to factors other than synthesis of HBV. In many patients with inactive viral infection a primary pathogenic factor was the HBV-associated delta, an agent with a putative RNA genome against which DNA inhibitors have no rationale and possibly no effects. The majority of Italian carriers do not appear suitable for treatment with DNA inhibitors and they should be considered for a different therapy.

Replication and amplification of recombinant plasmid molecules as extra chromosomal elements in transformed mammalian cells

Mouse thymidine kinase negative (TK −) L cells, F4-12B2TK − Friend erythroleukemic cells and hamster BHKTK − cells were transformed in the absence of carrier DNA with closed circular molecules of herpes simplex virus 1 TK DNA cloned in plasmid pAT153 (pTK-1). The physical status of donor DNA in the transformed cells was studied by Southern blot hybridization and spot hybridization techniques. Up to 65 copies of pTK-1 DNA molecules/cell were present in transformed cells grown under selective conditions. Whereas a steady increase in the number of pTK-1 copies/cell was found during the first few weeks of growth in selective medium, 2–8 months later copy numbers varied within the same cell line and their numbers rarely exceeded fifty copies/cell. Donor DNA sequences were found both in the Hirt precipitate and in the supernatant showing that some of the pTK-1 molecules existed in circular form. Many of the cell lines gave a Southern blot hybridization pattern indicative of pTK-1 sequences integrated into high molecular weight DNA as well as present in a circular configuration.