Comparison of DNA enzyme immunoassay and line probe assays (Inno-LiPA HCV I and II) for hepatitis C virus genotyping.

Two methods for genotyping hepatitis C virus (DNA enzyme immunoassay [DEIA] and line probe assay [Inno-LiPA HCV I and II]) were compared on 120 samples and of these 87% were assigned to the same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the core and 5' untranslated regions (UTR) were sequenced to resolve conflicts. Type 1 discordant samples had a guanosine at position -99 in the 5' UTR, a characteristic of genotype 1b, and a core region typical of subtype 1a. The eight isolates classified as 2a/2c by LiPA and as subtype 2c by DEIA belonged to type 2.

A DNA hybridization method for typing hepatitis C virus genotype 2c.

A high prevalence of hepatitis C virus (HCV) genotype 2c (22%) was detected in sera from 459 italian patients by core-region amplification and hybridization with specific probes by DNA enzyme immunoassay. Amplified fragments failed to hybridize with 1a, 1b, 2a, 2b and 3a subtype-specific and 4, 5, 6 type-specific oligonucleotides in 105 patients. Hybridization of these samples with type 2 probe, which recognized all the subtypes sequences, showed evidence for genotype 2 distinct from 2a and 2b. Fourteen out of these 105 isolates were cloned and sequenced. The results were consistent with genotyping assay. Nucleotide sequences were partially related to types 2a, 2b, 2d, 2e and 2f (87.0-93.5% of identity). The average nucleotide identity was highest for genotype 2c (95.87%). On the basis of sequence analysis, subtype 2c specific probe was derived. Hybridization efficiency with the newly designed probe was very high and more than 95% (100/105) of type 2 cases were classified as 2c. Evidence of different outcome of therapy inside the same HCV major type account for the need of accurate subtyping. In this study, amplification of the core region followed by hybridization with highly specific probes enabled distinction between HCV subtypes.

Detection of eight beta-thalassemia mutations using a DNA enzyme immunoassay.

We describe the use of a polymerase chain reaction-based method followed by a DNA enzyme immunoassay for the simultaneous detection of the eight most common beta-thalassemia mutations in the Mediterranean population. The method is specific, sensitive, and easily applicable in routine clinical laboratories for the molecular diagnosis of beta-thalassemia patients and at risk couples.

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

BACKGROUND: The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. OBJECTIVES: In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). STUDY DESIGN: The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. RESULTS: In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. CONCLUSION: This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

Detection of hepatitis D virus RNA in serum by a reverse transcription, polymerase chain reaction-based assay.

We designed a reverse transcription, polymerase chain reaction-based assay for serum hepatitis D virus RNA. Amplified hepatitis D virus cDNA was revealed by ethidium bromide staining, followed by blotting onto a nylon membrane and hybridization with a 32phosphorus-labelled oligonucleotide, or by a DNA enzyme immunoassay (DEIA) using a double stranded DNA-specific monoclonal antibody. The absolute sensitivity was expressed as number of hepatitis D virus RNA molecules, using a serum of known viral RNA concentration. Three sets of primers were used, encompassing the base positions 66-686 (variable rod-stabilizing region), 701-962 (conserved, viroid-like domain) and 886-1,333 (portion of the open reading frame 5 encoding for the carboxyterminus of the hepatitis D antigen) of the viral genome. The lower detection limits, after amplification of the three RNA portions, as assessed by ethidium bromide staining, were 7.5 x 10(6), 7.5 x 10(4) and 7.5 x 10(2) molecules of hepatitis D virus RNA per assay, respectively. The region encompassing bases 886-1,333 was chosen for blotting and hybridization to a radiolabelled oligonucleotide probe or for a capture-based DNA enzyme immunoassay, where the microplate was coated with this same probe. The two procedures showed comparable sensitivity, i.e., about 10 molecules of viral RNA per assay. The specificity of the assay was further on a panel of both anti-hepatitis D-positive and -negative sera. Amplification of serum hepatitis D virus RNA by reverse transcription/polymerase chain reaction followed by detection of the amplified cDNA by DNA enzyme immunoassay is a promising and feasible routine assay for detecting low amounts of circulating virions.

Simultaneous detection of delta F508, G542X, N1303K, G551D, and 1717-1G-->A cystic fibrosis alleles by a multiplex DNA enzyme immunoassay.

We describe the use of a polymerase chain reaction in conjunction with a DNA enzyme immunoassay for the simultaneous detection of five common cystic fibrosis mutations. The method is specific, sensitive, rapid, and proved effective in Guthrie card-based screening of cystic fibrosis mutations.

Typing of hepatitis C virus isolates by DNA enzyme immunoassay.

Recently, at least six types of hepatitis C viruses (HCV) have been identified. Different types of HCV appear to possess different pathogenic properties and a different sensitivity to interferon treatment. Typing of HCV isolates may therefore be an important diagnostic procedure. We report on a new method for identification of HCV types 1a, 1b, 2a, 2b and 3a which are most prevalent in Europe, North America and Japan. The assay is based on a combination of two well established techniques, the polymerase chain reaction (PCR) and DNA enzyme immunoassay (DEIA). In the first step of the method a cDNA of about 250 bp corresponding to the HCV core-region is amplified by nested PCR. The target cDNA is then hybridized to type-specific oligonucleotides fixed to a solid phase through an avidin-biotin bridge. The formed hybrids are detected by a standard ELISA using monoclonal antibodies reacting with double-stranded DNA. Typically, signal-to-noise (S/N) ratios between 18.2 and 48.6 could be observed when different HCV types/subtypes were analyzed by this method. The test was evaluated using cloned HCV cDNAs of known types and by sequence determination of some of the typed cDNAs. Typing of 115 isolates from Germany, Russia and Turkey revealed that subtype 1b (59-100%) and 1a (24-32%) are most prevalent in these countries.

A new method for direct analysis of polymerase chain reaction-amplified human papillomavirus using DNA enzyme immunoassay.

We describe use of a polymerase chain reaction in conjunction with a DNA enzyme immunoassay for the simultaneous detection of multiple types of human papillomavirus in a single microtiter plate. The method is specific, sensitive, rapid, and capable of detecting human papillomavirus-6, -11, -16, -18, and -33 in cervical biopsy specimens.

Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction.

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.

Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates.

The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein-Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.