Simultaneous amplification and detection of specific hepatitis B virus and hepatitis C virus genomic sequences in serum samples

A fluorescent antibody (FA) assay for hepatitis E virus antigen (HEVAg) in infected liver tissue was used to confirm the presence of virus-specific antigens in hepatocytes during the course of infection. With the cloning of the HEV genome it is now possible to determine which viral antigens are recognized by this FA assay. Recombinant HEV proteins covering the carboxyl half of HEV open reading frame 2 (ORF2) were used in this study to demonstrate that some of the most immunoreactive virus-specific antigens detected by FA are contained within this region of ORF2 (nucleotides 6169–7126).     

Detection of hepatitis B virus in serum using amplification of viral DNA by means of the polymerase chain reaction

A new assay was developed for the detection of hepatitis B virus (HBV) in human serum using amplification of a short viral DNA sequence by means of the polymerase chain reaction. As little as 0.4 fg viral DNA, corresponding to about 130 genome equivalents, per ml serum could be detected after the amplification procedure. This assay detected viral DNA in a number of patients with proven or suspected chronic HBV infection who were all negative for HBV DNA in the conventional hybridisation assay. We found HBV DNA in all of six HBeAg-positive and in three of eight HBeAg-negative HBsAg carriers, as well as in all of 11 patients with chronic liver disease with antibodies against the HBV core antigen (anti-HBc) as the sole marker for HBV infection, and in three of five apparently healthy individuals showing only anti HBc. Thus, this method is an important improvement for the diagnosis of persistent HBV infections, especially in patients where a definitive serological diagnosis is not possible.     

应用扩增受阻突变检测系统（ARMS）检测HBV基因型

目的建立扩增受阻突变检测系统（ARMS）检测乙型肝炎病毒（HBV）基因型方法,并对慢性乙型肝炎（CHB）患者HBV基因型进行分析。方法通过对HBV全基因组序列比对分析,设计ARMS特异性引物和探针体系,建立检测HBVB和C基因型方法,对50例临床标本进行检测。结果50例临床样本中,B型的检出率为26％（13例）,C型为74％（37例）,其实验结果与测序结果完全一致。常州及周边地区乙型肝炎病毒C基因型HBV为优势株。C基因型患者A1762T／G1764A突变率为62％,明显高于B型。结论ARMS检测HBV基因型,快速、准确;常州及周边地区HBV基因型主要为B、C型,且C型携带A1762T／G1764A双突变率较高,与较为严重的肝病相关。     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization.

We have developed a microtiter sandwich hybridization assay for the detection of polymerase chain reaction (PCR)-amplified hepatitis B virus (HBV) sequences. This assay utilizes an enzyme-linked immunosorbent assay-like format in which cloned DNA containing a sequence complementary to half of one PCR product strand is immobilized in microtiter wells. A biotin-labeled DNA sequence complementary to the other portion of the same PCR product strand is used as the probe. The DNAs from 69 hepatitis B surface antigen-positive serum samples and 16 antigen-negative control samples were amplified by the PCR procedure, and the product was detected by Southern and sandwich hybridization. Both detection procedures were capable of detecting as few as five copies of HBV DNA. Compared with Southern hybridization, the sandwich hybridization assay exhibited a sensitivity of 100% and a specificity of 95% for the detection of amplified HBV sequences. Unlike Southern hybridization, however, the sandwich hybridization assay employs a nonradioactive probe and allows easy handling of large numbers of samples. DNA was detected in 74% of the antigen-positive samples. All of the antigen-negative samples (healthy blood donors) were negative for HBV DNA by both procedures.     

Detection of hepatitis B virus DNA in serum using synthetic non-radioactive oligonucleotides.

A rapid and simplified technique for detecting hepatitis B virus (HBV) DNA by spot hybridisation in the sera of patients with different clinical forms of HBV infection was investigated using enzyme conjugated synthetic oligodeoxyribonucleotides as probes. These are able to hybridize to the S and C regions of the HBV L(-) DNA strand. When compared with a complete 32P-labelled HBV DNA probe, the synthetic oligonucleotides provided a sensitive and quick method for the routine survey of HBV infection. Moreover, the DNA extraction procedure used allowed the spot hybridisation technique to be applied and read easily and the results obtained within a few hours. It is concluded that synthetic cold probes can be used in hybridisation assays HBV DNA detection as part of current clinical laboratory procedures.     

Detection of serum hepatitis B virus DNA using the polymerase chain reaction assay

We compared the sensitivity of the polymerase chain reaction (PCR) assay to that of slot blot hybridization for detecting hepatitis B virus (HBV) DNA in the serum of a chimpanzee infected with HBV and 52 patients. Also, we utilized a rapid PCR procedure for the detection: Viral DNA was released from virions by incubating serum with NaOH. After a primary PCR amplification, the sample was reamplified using a second set of primer pairs (PCR/PCR). In the chimpanzee, HBV DNA was detected 3 weeks earlier than the appearance of hepatitis surface antigen (HBsAg) and persisted for two weeks with antibody to HBsAg. Of the 14 chronic hepatitis B patients positive for both HBsAg and HBV e antigen (eAg), 9 were positive for HBV DNA by slot blot hybridization and all 14 by PCR. Also, of 9 patients positive for HBsAg and antibody to eAg, 2 were positive for HBV DNA by slot blot hybridization and 8 by PCR. Three of the 11 patients who had lost HBsAg during follow up examination of chronic hepatitis B were positive for HBV DNA by PCR, whereas none of them was positive by slot blot hybridization. Six patients who had recovered from acute hepatitis B more than one year ago and 12 cases who had had vaccination of HBV were negative for HBV DNA by PCR. This technique should yield valuable information on the biology of HBV.     

Detection of hepatitis B virus DNA in serum by a rapid filtration-hybridization assay

A simple method for detecting hepatitis B virus DNA (HBV DNA) in serum using a filtration step for spotting sera on nitrocellulose paper followed by molecular hybridization is described. This method is rapid, sensitive, requires very small quantities of serum, and can be used for simultaneous testing of up to 96 samples in one filter apparatus. The sera tested for HBV DNA were also assayed for serological markers of HBV infection and comparison of data shows that on average 67% (30 of 45) of HBsAg- and HbeAg-positive sera contain HBV DNA, whereas 13% of HBsAg- and anti-HBe-positive sera contain HBV DNA. In general, there was a statistically significant correlation between the concentration of HBsAg in the serum and the presence or absence of HBV DNA. These results indicate that molecular hybridization is a valuable assay in addition to serological markers for identifying the possible infectivity of sera, and the simple and rapid method reported here makes the use of such hybridization technique easier.     

Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR)

Summary The Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). The sequence selected for amplification consists of 238 bp lying in the gene encoding the fusion protein F. A pair of 19-mer and 18-mer oligonucleotides, flanking this sequence, were used as primers. Following RNA extraction by the proteinase K method, a cDNA was prepared using the previous 19-mer oligonucleotide as the primer. The amplification reaction product was analyzed by electrophoresis and ethidium bromide staining, using the restriction enzymes Hae III, Mbo II, and Nar I. The PCR was performed on cDNA prepared from 30 A-PMV 1 and 3 other strains (A-PMV 2, A-PMV 3, A-PMV 4). It was thereby demonstrated that the selected sequence was highly specific and constant. However, two of the PMV 1 strains isolated from feral ducks, are thought to present a deletion of about 25 bp inside this fragment as shown by the smaller length of the corresponding amplified product and the disappearance of the Nar I restriction site.The advantages of this technique, as a first step in evaluating virulence by means of molecular biology, is discussed.     

In vitro amplification of hepatitis B virus sequences from liver tumour DNA and from paraffin wax embedded tissues using the polymerase chain reaction.

A 185 base pair fragment from the core-polymerase overlap region of the hepatitis B virus (HBV) genome was amplified using the polymerase chain reaction (PCR). The results were compared with those of Southern blotting on extracted DNA from eight hepatocellular carcinomata. The data agreed with those of Southern blotting in six cases (two positive, four negative) but in two other positive cases PCR failed to amplify HBV sequences. This suggests deletion or mutation, or both, of this viral region in these cases. PCR was also used to amplify HBV sequences from formalin fixed, paraffin wax embedded tissue. Tissue inhibition of PCR occurred which increased with the number of tissue sections. It was present in tissues from different organs and species and fixed by different procedures, thus highlighting the need for a positive control during amplification. Use of formalin fixed Alexander cells, however, showed a sensitivity of one viral copy per 5000 cells. Confirmation of the identity of the PCR products was carried out using PCR-generated biotinylated probes, and suggested the insertion of extra nucleotide sequences or infection with an HBV variant in one case.     

Comparison of methods for extraction of nucleic acid from hemolytic serum for PCR amplification of hepatitis B virus DNA sequences.

The sensitivity of PCR for the amplification of target nucleic acid sequences in clinical diagnostics may often be reduced due to the presence of inhibitory factors. Hemolytic serum contains a number of PCR inhibitors, one of which is hemin. In this study we have found that conventional methods of DNA extraction were not sufficient for the removal of PCR-inhibitory compounds in hemolytic serum. We have therefore compared the efficiency of several commercial and noncommercial methods of nucleic acid purification from hemolytic serum samples prior to PCR amplification. Separation with the QIAamp HCV kit, dialysis with Millipore filters, and bovine serum albumin absorption were all found to be suitable extraction methods for eliminating inhibitors from hemolytic serum for PCR amplification. Using these methods we were able to detect very low levels of hepatitis B virus DNA in hemolytic serum.     

Detection of hepatitis B virus DNA in serum and relation with the IgM class anti-HBc titers in hepatitis B virus infection

Sera from four groups of patients wtih different serologic markers of HBV infection were examined for HBV DNA using molecular hybridization technique and for IgM class anti-HBc using an ELISA based on the antibody capture principle. Results of HBV DNA assay were generally in good agreement with the presence of HBeAg. However, HBV DNA was found in 13% of anti-HBe+ sera and in one patient with anti-HBc as a sole marker. IgM anti-HBc was detected at high titers in acute hepatitis B patients and was also present during the "window-period." This marker was also found, though less frequently when other markers for HBV infectivity were absent, in chronic hepatitis B patients and healthy carriers. From these findings we conclude that the HBV DNA assay provides a reliable method of detecting the infectious agent, particularly in anti-HBe+ sera and sera with anti-HBc as a sole marker. The assay for IgM anti-HBc is useful for establishing the diagnosis of recent infection in patient with anti-HBc as a sole marker, and during acute hepatitis with very high aminotransferase values, a condition in which HBV DNA may be undetectable.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR）,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳,EB染色,UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在10^5/ml以上者为阳性,用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定性PCR观察到193例有阳性特异带,阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便,灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点,值得在临床检验中推广应用。     

不同慢性肝病患者血清中乙型肝炎病毒DNA定量检测及其临床意义

目的探讨乙型肝炎病毒ＤＮＡ含量的临床意义。了解乙型肝炎病毒（ＨＢＶ）免疫标志不同状态的慢性肝病患者血清ＨＢＶＤＮＡ浓度及其临床意义。方法应用建立的竞争性聚合酶链反应（ＰＣＲ）方法定量检测慢性肝炎（ＣＨ）５１例、肝硬化（ＬＣ）３６例、原发性肝癌（ＰＨＣ）３８例的血清ＨＢＶＤＮＡ浓度。结果ＨＢＶＤＮＡ阳性的ＣＨ患者血清ＨＢＶＤＮＡ浓度为４．３６ｌｏｇ１０ＨＢＶＤＮＡ拷贝５０μｌ（下同），ＬＣ为４．５５，ＰＨＣ为４．４３，三组间无显著性差异（Ｐ＞０．０５）；血清ＨＢＶ五项免疫标志均阴性或抗－ＨＢｓ阳性的慢性肝病患者中，有３７．５％患者存在低水平ＨＢＶ复制；ＨＢｅＡｇ阳性患者的ＨＢＶＤＮＡ浓度总体上明显高于抗－ＨＢｅ阳性组，但其中部分患者的ＨＢＶＤＮＡ浓度也很高。结论提示ＨＢＶ的复制状态与慢性肝病的病期无明显关系；在抗－ＨＢｅ阳性的患者中存在个体差异，故不能仅依据抗－ＨＢｅ阳转来判断ＨＢＶ复制减少或停止。     

Detection of hepatitis B virus DNA in the serum of Canadian hepatitis B surface antigen negative,anti-HBc positive individuals,using the polymerase chain reaction

Continuing hepatitis B virus (HBV) infection is normally associated with the presence of hepatitis B surface antigen (HBsAg) in the serum. In spite of sensitive screening assays for HBsAg, rare cases of post-transfusion HBV infection are still observed. Antibody to hepatitis B core antigen (anti-HBc) often indicates remote HBV infection but DNA hybridisation and more sensitive polymerase chain reaction (PCR) assays have demonstrated that some HBsAg negative individuals, positive for anti-HBc, have continuing HBV replication. To determine the incidence of ongoing HBV infection in a Canadian HBsAg negative, anti-HBc positive population we studied three groups with this combination of HBV markers: Group A, 36 patients referred for investigation of raised serum aminotransferases; Group B, 21 Canadian Red Cross blood donors; Group C, seven vaccinees in an Ottawa Health Care Student hepatitis B vaccination programme. The PCR was carried out using a nested PCR reaction with primers specific for the pre-core region of HBV. Seven of 36 (19%) patients in Group A had detectable HBV DNA whereas none of Group B or C were positive. This data indicates that in some HBsAg negative patients with ongoing hepatic inflammation, continuing HBV replication may persist. This was not observed in any healthy blood donors or health care students investigated. Larger studies are required, but this data would suggest that, in Canada, the addition of anti-HBc testing for all blood donors for detection of low level HBV replication would not be indicated. © 1994 Wiley-Liss, Inc.     

Detection of hepatitis B virus DNA in sera from patients with chronic hepatitis B virus infection by DNA microarray method

We have developed a sensitive and quantitative assay using a DNA microarray for the detection of hepatitis B virus (HBV) DNA in serum. Fluorescently labeled target cDNA prepared from cloned HBV DNA or serum HBV DNA was hybridized to capture DNA on a slide. A linear relationship was obtained between the intensities of the array spot and the amount of the cloned or serum HBV DNA, indicating the quantitative accuracy of this assay system. In addition, there was a significant correlation between the number of molecules of serum HBV DNA determined by the DNA microarray and that determined by a branched-DNA assay (n = 21, r = 0.89). Given these results, we conclude that the DNA microarray assay system may be useful as a diagnostic technique in the clinical laboratory.     

Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay

The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01?ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3)?IU/mL (χ(2)?=?25.04, p?相似文献