HBV-DNA Probe Research Articles

A microfluidic device with microbead array for sensitive virus detection and genotyping using quantum dots as fluorescence labels

In this study, a novel microfluidic device with microbead array was developed and sensitive genotyping of HBV was demonstrated using quantum dot as labels. This device was assembled by using two PDMS slabs featured with different microstructures and channel depths for the construction of a functional region comprising a chamber array and a single sampling microchannel. Since the chamber array and its sampling channel are of different channel depths and are bonded face-to-face, weir structures are generated to confine the microbeads which could be addressed using the microfluidic channel. Highly sensitive virus DNA detection was achieved by the enhanced mass transport in the microfluidics and the rapid reaction dynamics of suspension microbead array. The device could detect 1000 copies/mL of HBV virus in clinical serum samples using in vitro transcribed RNA as the target molecules. Based on DNA hybridization with quantum dots labels, on-chip virus genotyping was also demonstrated with high discrimination specificity and sensitivity (4 pM, S/N >3) using synthesized HBV DNA probes. This microfluidic device combines the rapid binding kinetics of homogeneous assays of microbead array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for high sensitivity virus DNA analysis with small reagent consumption, short assay time and parallel detection.

Detection of HBV and HCV coinfection by TEM with Au nanoparticle gene probes

Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy (TEM). PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection was added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes. The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.

Measure Hepatitis B Patient Serum of HBV DNA Content and Probe into Their Clinical Meanings

目的 荧光定量PCR检测乙肝患者血清的HBVDNA含量,并探讨及其对临床意义.方法对520例怀疑乙肝患者的血清进行ELISA法检测乙肝病毒血清标志物(HBVM)和荧光量聚合酶链反应(FQ-PCR)检测HBVDNA的含量并进行比较.结果检测的520例血清标本中HBVDNA水平在不同乙型肝炎类型中显著性差异(P＞0.05),与HBV-M主要阳性模式组对比,在135阳性组HBVDNA的阳性率为97.7%(164/168),1 3模式组阳性率为100%(20/20),145模式组阳性率为55.56%(15/27),在检测的全阴性的模式组中有一例DNA阳性为5.88%(1/17).结论HBVDNA的含量在不同的阳性模式中均不同程度地检测出HBVDNA的含量.其中,HBeAg阳性血清标志物中HBVDNA的检测率最高几乎达到100%.采用简单快捷的荧光定量PCR检测对乙肝患者的带毒状态进行准确判断.但HBVDNA的复制状态与不同临床类型乙型肝炎无明显相关关系,HBVDNA含量高低与肝功能的损害程度亦无明显关系。

The application of alkaline phosphatase labeled HBV probe in serum detection.

A method using non-radioactive material alkaline phosphatase to label HBV DNA as probe has been studied and used in clinical experiments to detect the HBV DNA in hepatitis serum. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as cross-linking reagent. The modified phosphatase was covalently linked to single strand DNA using glutraldehyde. Such single strand DNA enzyme complexes have been tested for blot hybridization, after hybridization and incubation with a substrate solution, sequences complementary to the probe can be visualized directly in 1 h. The minimum amount about 10 pg of target DNA has been detected in this way, 32P labeled probes are autoradiography 1 h after hybridization can only detect 10 ng, so the enzyme labeled probe is more sensitive than isotope labeled probe in 1 h fast test. Comparing the enzyme-labeled HBV DNA probe with 32P labeled the same one, positive proportion of detecting the HBV DNA in hepatitis patients was about 95.7%. Because the positive patient's serum detected by 32P labeled probe were selected through 1- week radiation, Alkaline Phosphatase labeled probes are color developed for only 1 h. Our experiment certified that it is a sensitive, specific, easy, rapid, safe and economical probe labeling and clinical virus DNA detection method.

Effects of hepatitis B virus infection on human sperm chromosomes.

To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients. Sperm chromosomes of 14 tested subjects (5 healthy controls, 9 patients with HBV infection, including 1 with acute hepatitis B, 2 with chronic active hepatitis B, 4 with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. The total frequency of sperm chromosome aberrations in HBV infection group (14.8 %, 33/223) was significantly higher than that in the control group (4.3 %, 5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4 signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random. HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice.

Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transferring the Simian Virus 40 Large T-Antigen into primary human hepatocytes. These cells were stably transfected with a full-length HBV genome to generate a clone that expresses HBV genes and replicates HBV. The HBV-producing cells were transplanted into the livers of mice with combined immunodeficiency (Rag-2 deficient) by intrasplenic injection. Survival of the engrafted human hepatocytes was shown in several ways: fluorescent in situ hybridization (FISH) with a human-chromosome-specific DNA probe (human alpha satellite), dot-blot hybridization of the genomic DNA extracted from liver biopsy specimens with a human-specific Alu repetitive DNA probe, Blur-8, as well as with an HBV DNA probe, and secretion of human proteins into plasma. Histological examination of mouse liver up to 8 months following human cell transplant shows completely normal architecture. Determination of plasma HBV DNA levels indicated that engrafted cells secreted 3x10(7) to 3x10(8) virions per mL into the blood, and HBsAg was detected in plasma. This new murine model of HBV viremia should be useful for in vivo HBV studies.

HBx-DNA probe preparation and its application in study of hepatocarcinogenesis.

AIM:To study the role of HBV especially HBx Open Reading Frame (ORF) in the development of hepatocellular carcinoma (HCC).METHODS:HBV 3.2kb fragment was retrieved by digesting recombinant plasmid pBR(322)-2HBV with EcoR II, and HBx 0.59kb fragments by digesting HBV-DNA with BamH I and Bgl II. These fragments were labelled with digoxigenin to get HBV-DNA and HBx-DNA probes. HBV-DNA was detected in HCC by dot blot and Southern blot hybridization with HBV-DNA probe, so the positive specimens in which HBV-DNA were integrated were selected. HBx-DNA was subsequently detected in the selected specimens with HBx-DNA probe.RESULTS:HBV-DNA was detected in 75% HCC, among which integrated type, integrated + free type covered 63.6% and 36.4%. There was no free type. HBx-DNA was detected in 90.5% specimens of integrated type.CONCLUSION:Hepatocarcinogenesis was highly related to HBV-DNA integration, and HBV-DNA mainly integrated into chromosome with incomplete virus DNA fragments among which HBx fragment was the predominant one.

Three distinct Southern blot hybridization patterns of HBV-DNA in the sera of HBV carriers.

The molecular species of hepatitis B virus (HBV)-DNA of 44 sera taken from 25 HBV carriers were examined by Southern blot hybridization with a biotin-labeled HBV-DNA probe and classified into three patterns. Type I consisted of two distinct bands of 4.0 kb and 3.2 kb. Type II consisted of the 4.0 kb and 3.2 kb bands and a smear between these two bands. Type III showed a broad band below 4.0 kb. With endogenous HBV-DNA polymerase reaction, the smear disappeared and 4.0 kb and 3.2 kb bands appeared. After EcoRI digestion, the 4.0 kb band disappeared to form a single band of 3.2 kb. These results suggest that the 4.0 kb and 3.2 kb forms are a relaxed circular, fully double-stranded DNA and a linear, fully double-stranded DNA, respectively, and that the smear between 4.0 kb and 3.2 kb is formed from the sum of relaxed circular, partially double-stranded DNAs with various length of plus strands. Comparison between histological diagnosis and Southern blot hybridization patterns of 25 HBV carriers indicates that these three patterns are closely related to the degree of hepatitis.

Detection of hepatitis B virus DNA by polymerase chain reaction in HBsAG negative Senegalese patients suffering from cirrhosis or primary liver cancer

The polymerase chain reaction was used to search for hepatitis B virus (HBV)-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer. Amplified HBV-DNA sequences were detected by hybridization with a digoxigenin-labelled HBV-DNA probe. HBV-DNA was detected in 17% of HBsAg negative Senegalese subjects from the general population and in 44% and 58% of the patients suffering from cirrhosis or primary hepatocellular carcinoma (PHCC) respectively. In the control group, amplified HBV-DNA was detected in 25% of the subjects without HBsAg and anti-HBs antibodies, and in 6% of subjects positive for anti-HBs antibodies. This study confirmed the hypothesis that there is an etiologic link between HBV and PHCC in HBsAg-negative patients.

Detection of HBV-DNA using a digoxigenin-labelled probe

The sensitivity of four different non-radiolabelled HBV-DNA probes (digoxigenin-, biotin-, acetylaminoflorene-, chemiprobe-labelled) in detecting HBV-DNA were compared with a 32P-labelled HBV-DNA probe using a spot hybridisation assay. Of the non-radiolabelled probes, the probe using digoxigenin labelling was the most sensitive with reliable and reproducible detection to between 1-3 pg HBV-DNA. The use of digoxigenin-labelled HBV-DNA hybridisation assay deserves wider application for the detection of HBV-DNA because of a number of advantages including ease, speed, safety and cost.

IN SITU HYBRIDIZATION FOR DETECTION OF HEPATITIS B VIRUS GENOMES IN LIVER TISSUE OF CHRONIC INFECTED CHILDREN

Detection of hepatitis B virus (HBV)-DNA in the liver of chronic infected patients is presently the most sensitive marker of viral replication and infectivity. In situ hybridization (ISH) allows the direct visualization of HBV infected liver cells and distribution of the viral sequences. This study was done to establish ISH and correlate the findings with conventional markers for HBV infection. Methods. Liver biopsies of 50 patients (28 ♂, 22 ♀) aged 0.5-20 years (mean 10.3) with various histological diagnoses were tested by 1SH. The HBV-DNA probe was labeled by nick translation with 35S-CTP to a specific activity of 3-5×108 cpm/μg DNA. Results. HBV-DNA/mRNA could be demonstrated in 38 patients, 12 were negative. Distribution of the grains was homogenous, inhomogenous with focal patches and focal. 33/38 children with HBeAg and 4/11 with anti-HBe were positive by ISH, 5/38 with HBcAg and 7/11 with anti-HBe remained negative. 17/23 HBsAg carriers with positive HBV-DNA/mRNA by ISH were positive for MBcAg in the liver and 31/36 had free HBV-DNA in Southern blot hybridization. Conclusions. Our results indicate that hepatitis B viral genomes can reliably be detected by in situ hybridization. Although there is a good correlation to other HBV markers (HBcAg, HBcAg and Southern blot hybridization) ISH may represent a more sensitive method to prove viral replication and infectivity.

Serum hepatitis B viral DNA in HBsAg-positive hepatocellular carcinoma treated with interferon or adriamycin.

Sera from 31 HBsAg-positive Chinese patients with inoperable hepatocellular carcinoma (HCC) were tested for hepatitis B virus DNA (HBV DNA) by means of dot hybridisation and Southern blot technique. HBV DNA probes were prepared from human plasma. Eighteen of the patients were HBeAb-positive, 12 were HBeAg-positive and one case had neither marker. Serial specimens were obtained from 16 cases over 5-42 weeks, while the patients were treated with recombinant leukocyte A interferon (rIFN-A) or adriamycin. Seven patients (2 HBeAg-positive, 5 HBeAb-positive) were positive for HBV DNA. In two patients HBV DNA and HBV DNA polymerase (DNAp) appeared in serum weeks after rIFN-A or adriamycin treatment was started. In two other cases, HBV DNA that was initially present disappeared during rIFN-A treatment. In a fifth patient HBV DNA persisting after adriamycin treatment diminished after change of treatment to rIFN-A. With one possible exception the HBV DNA detectable by Southern blot technique was composed chiefly of sequences 2.2-3.2 kb size indicating the presence of unintegrated DNA forms. DNAp activities were raised in the presence of HBV DNA in 4 patients. These findings show that HBV replication can be activated or suppressed in advanced HCC. Treatment with rIFN-A may have been effective in suppressing HBV DNA synthesis, but the number of cases studied was too small to arrive at a definite conclusion on this point.

Search for hepatitis B virus DNA in sera from patients with acute type B or non-A, non-B hepatitis

One hundred and three single sera from adults hospitalized with acute type B (78) or non-A, non-B (25) hepatitis were tested for the presence of hepatitis B virus DNA (HBV DNA). All sera from patients with type B hepatitis were IgM anti-HBc-positive. These patients were classified as benign (47) or fulminant (31) hepatitis. The 25 acute non-A, non-B patients were also classified as benign (21) or fulminant (4) hepatitis and were negative for serologic markers of past HBV infection. Serum HBV DNA was detected with similar frequency in benign (38.5%) and fulminant (FH, 34.6%) HBsAg-positive cases. HBV DNA was not detected in either the 26 acute HBsAg-negative hepatitis B cases who were positive for anti-HBc and anti-HBs or the 25 acute non-A, non-B hepatitis cases. The absence of HBV DNA in 43.8% of benign hepatitis B patients who were positive for HBsAg and HBeAg could possibly be attributed to either low level replication of HBV that was not detectable by the [32P]HBV DNA probe or to a period of delayed clearance of free HBeAg following cessation of HBV replication. Emergence of anti-HBs in the presence of HBsAg did not always correspond to clearance of HBV in fulminant type B cases. However, in acute type B hepatitis, irrespectively of severity, disappearance of HBsAg and appearance of anti-HBs was accompanied by reduction of HBV replication to undetectable levels.

The detection of HBV-DNA in serum by molecular hybridisation: a more sensitive method for the detection of complete HBV particles.

Existing methods for detecting complete virus particles in the serum of patients with chronic HBV infection are either insensitive or indirect. A method is described in which Dane particle-associated DNA is extracted from a small volume of serum and detected by molecular hybridization using 32P-labeled cloned HBV-DNA or HBV-DNA extracted from the serum of an immunosuppressed patient, followed by autoradiography and densitometry. There was a positive correlation between the amount of HBV-DNA detected using HBV particle-derived and cloned HBV-DNA probes. The amount of HBV-DNA detected in serum samples showed a positive correlation with the HBV-DNA polymerase. The method was more sensitive than the DNA polymerase and HBeAg assays in detecting complete virus particles. It may be useful in determining the level of infectivity in patients and in monitoring response to antiviral therapy.