Quantification of hepatitis B virus DNA over a wide range from serum for studying viral replicative activity in response to treatment and in recurrent infection.

A new standardized test for hepatitis B virus (HBV) DNA with increased sensitivity and range over previous assays (30 to 10(6) HBV genomes/test) was evaluated in this study. The quantitative results from the test have been validated using international reference specimens of known titer and a reference solution hybridization test. The test has small variability considering the wide dynamic range. The CV was 14% within one experiment and 32% to 39% between independent experiments. Hepatitis B surface antigen (HBsAg)-negative, anti-HBc-positive blood donor sera (n = 25) were all negative for HBV DNA in the new test, whereas 63% (n = 19) of HBsAg-positive healthy carriers had measurable quantities of HBV DNA. In five example cases of chronic hepatitis B patients responding to alfa-interferon treatment but remaining virus positive, HBV DNA was consistently present in posttreatment sera in a titer range 4 x 10(3) to 10(6)/mL not detectable by the conventional hybridization test. In two complete responders, the HBV DNA titer decreased over six orders of magnitude to below cutoff of the test. In four liver transplant recipients with chronic hepatitis B, viral recurrence was detected by the new test at an early stage much before the clinical relapse. Unlike serology, the test was suitable also in patients under anti-HBs immunoprophylaxis. In conclusion, the new colorimetric polymerase chain reaction (PCR) test allowed thousandfold increased sensitivity in quantification of HBV DNA in patient sera. The test may have future applications in improving assessment of efficacy of antiviral treatment and guiding therapeutic interventions.

Quantitative analysis of wild-type and HBeAg minus hepatitis B viruses by a sequence-dependent primer extension assay.

The ratio between wild-type hepatitis B virus (HBV) and HBV mutant, unable to secrete "e" antigen (HBeAg minus HBV) appears to be an important determinant of the outcome of chronic hepatitis B. Quantitative analysis of wild-type and HBeAg minus HBVs in the blood could be useful to monitor chronic hepatitis B patients. We developed a solid-phase minisequencing assay for both viruses using a primer-guided incorporation of a single labeled nucleotide on an affinity captured biotinylated amplified HBV-DNA template. A standard curve was constructed by mixing increasing quantities of wild type and mutant virus DNAs. The detection of wild-type and HBeAg minus sequences, ranging from 10% to 90% of overall viremia, was linear and reproducible till 0.1 pg/microliter of serum HBV-DNA. The assay yields numerical values and the ratio of incorporated nucleotides defines the relative proportions (%) of the two viral sequences with accuracy. We tested the sensitivity and accuracy of the minisequencing on mixed end point dilutions of wild-type and HBeAg minus reference sera and amplified products. The feasibility and reproducibility of the assay were tested in 35 sera from 21 HBsAg positive patients with chronic hepatitis B using both minisequencing and oligo-hybridization assays. A high correlation was found between the two assays (r = 0.957 P < 0.0001). In conclusion, the minisequencing assay provides a precise and reproducible quantitative analysis of wild-type and HBeAg minus HBVs in clinical specimens. It is proposed to study the relations between HBV heterogeneity and the course of hepatitis B and its response to therapy.

Detection of human papillomavirus DNA by AffiProbe HPV-DNA test kit in cervical scrapes or biopsies-histopathologic correlates.

OBJECTIVE: The aim of this study was to evaluate and compare the efficacy of punch biopsies and cervical scrapes in the detection of human papillomavirus (HPV) DNA from the cervix and compare the results with the histopathologic diagnosis. METHODS: The specimens were collected simultaneously, and HPV DNA was detected using a liquid hybridization test. RESULTS: Biopsies and scrapes were equally efficient, but each detected only two-thirds of all HPV-DNA-positive patients. Thus, the positivity rate increased when both tests were used. Overall, 13% of patients with normal histopathology, 38% of patients with benign atypia, and 66% of patients with squamous intraepithelial lesions (SIL) were HPV-DNA positive. HPV-DNA 16 was found in 54% of HPV-DNA-positive patients with SIL, in 20% of HPV-DNA-positive patients with atypia, and in none of patients with normal histopathology. CONCLUSIONS: The liquid hybridization test used in this study detects HPV DNA equally efficiently from both biopsies and scrapes. The test can be performed in 1 working day. However, the sensitivity of the test is low, and it only detects a limited number of HPV types.

Quantitative PCR for hepatitis B virus with colorimetric detection.

A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.

Colorimetric solid-phase minisequencing assay illustrated by detection of alpha 1-antitrypsin Z mutation.

In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Human papillomavirus detection from the female genital tract: combined vs. separate scrape methods.

Because genital human papillomavirus (HPV) infections tend to be multifocal, it was studied how effective one combined specimen is in detecting HPV-DNA from the lower female genital tract. The study population consisted of 50 patients referred to a colposcopy clinic for a suspected condylomatous and/or dysplastic lesion. From half of the patients, a separate scrape from the cervix, vagina and vulva was taken first followed by a combined scrape representing all the genital sites, and from the other half, vice versa. HPV-DNA (types 6, 11, 16 and 18) was identified using the AffiProbe hybridisation test. Thirty-six specimens collected from 17 patients were positive for HPV-DNA. A multifocal infection was demonstrated in at least 11/17 (65%) HPV-DNA positive patients. The combined scrape was the most informative specimen, revealing 75% of all HPV-DNA-positive patients. It was concluded that HPV-DNA can reliably be detected from the female genital tract in a simple way from one combined specimen.

A rapid and quantitative solution hybridization method for detection of HBV DNA in serum.

A sensitive and convenient solution hybridization technique was adapted for the semiquantitative detection of hepatitis B virus DNA in serum. The assay utilizes 35S-isotope as label and biotin-avidin interaction for collection of the hybrids onto microtitre plate wells. Results are obtained as numerical values, which allow quantification. 10(6) molecules of HBV DNA/ml serum could be detected by a 3-h hybridization followed by a 2-h collection reaction. By analyzing 500 microliters of serum, 30 (88%) of 34 patient sera positive for HBeAg were also positive for HBV DNA and 17 HBsAg-positive sera, 16 of which were anti-HBe-positive, were DNA-negative. The amount of HBV DNA varied from 5 x 10(6) to 3 x 10(9) molecules/ml. The solution hybridization method which was developed allows fast and accurate quantification of HBV DNA in serum providing an estimate of the virus titre.

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test.

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.

Screening for defined cystic fibrosis mutations by solid-phase minisequencing.

We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.

New microbial diagnosis.

Rapid methods for comprehensive nucleic acid analysis are essential for the progress in basic research. In microbial diagnosis nucleic acid analyses are useful for achieving quick, sensitive results with new dimensions of specificity. As to specificity, hybridisation tests are easily constructed for genus or species specific microbial typing or for the detection of genes accounting for pathogenic properties. Modern sequence analysis allows rapid detection of mutations eg., those conferring antiviral resistance. A new level of sensitivity comparable to that of the conventional enrichment culture is obtainable by enzymatic amplification (PCR) of microbial nucleic acids before detection. PCR-technology has proved particularly useful in the diagnosis of microbial infections when the organisms are impossible or tedious to cultivate. For reliable diagnostic use the performance of in vitro amplification requires extreme care to prevent false positive results owing to carry over problems. In conclusion, nucleic acid analyses are expanding the potential for diagnosing microbial infections and may well replace some conventional diagnostic methods.

A rapid solution hybridization method for detection of human papillomaviruses.

Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures. We have previously described the sandwich hybridization method which allows convenient testing of biological specimens. In this paper we describe the adaptation of the solution hydridization method into the microtitre plate format using 35S-isotope as label. Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 x 10(5) target DNA molecules was achieved. The method was applied for identification of human papillomaviruses in crude gynaecological specimens. A simple 1-day assay protocol was achieved with high HPV type specificity. The specificity was confirmed by testing a variety of unrelated micro-organisms, none of which gave a positive signal in the test. Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals.

Use of AffiProbe HPV test kit for detection of human papillomavirus DNA in genital scrapes.

The presence of human papillomavirus (HPV) DNA in cervical and vaginal scrapes was analyzed by the AffiProbe HPV test kit (Orion Corp., Orion Pharmaceutica, Helsinki, Finland), which is a 1-day solution hybridization test for HPV type 6/11, 16, or 18. The AffiProbe test was compared with a commercially available dot blot test (ViraPap and ViraType tests; Life Technologies Inc., Gaithersburg, Md.). The study group consisted of 178 patients seen in a gynecological outpatient clinic. Altogether, 64 specimens (36 cervical and 28 vaginal scrapes) from 49 patients were positive by the AffiProbe test. Concurrently collected cervical scrapes from 174 patients were available for the reference test, which yielded 27 positive results for HPV type 6/11 or 16/18 and 25 positive results for HPV type 31/33/35. Agreement as to the presence of HPV type 6/11, 16, or 18 by the two tests was reached in 85% of the specimens. Eleven cervical specimens were positive by the AffiProbe test only, and nine cervical specimens were positive by the ViraType test only. Independent evidence obtained by the polymerase chain reaction, repeat examination, or the concurrent presence of HPV DNA in vaginal or vulval epithelium supported the AffiProbe and the ViraType test results for 6 of the 11 and 6 of the 9 specimens with discrepant results, respectively. Thus, the DNA tests had similar sensitivities for HPV type 6/11, 16, and 18 DNAs, but the results were obtained within 1 day by the AffiProbe test, whereas results for the ViraPap and ViraType analyses required from 4 days to 2 weeks.

Affinity-based collection of amplified viral DNA: application to the detection of human immunodeficiency virus type 1, human cytomegalovirus and human papillomavirus type 16.

We have devised a sensitive and convenient hybridization technique by combining the polymerase chain reaction (PCR) with affinity-based hybrid collection. In this method 5'-biotinylated primers are used to introduce biotin residues into the DNA fragments during the amplification. The amplified DNA fragments are detected by liquid hybridization using a 32P- or 35S-labelled oligonucleotide as probe. For measurement the hybrids are collected on polystyrene microparticles or onto microtitre wells taking advantage of the biotinavidin interaction. The method is highly sensitive allowing the detection of 30 molecules of DNA. It involves few and simple operations, and is thus suitable for routine diagnostics. The applicability of the method to the detection of HIV-1 DNA from blood, HCMV DNA from urine and HPV-16 DNA from cervical scrapes was evaluated.

Sandwich hybridization in solution: a rapid method to screen HPV 16 DNA in cervical scrapes.

A solution hybridization method is introduced as a rapid diagnostic method for demonstration of papillomavirus DNA in cervical scrapes. 32P-Labelled detector probe and the biotinylated capture probes were hybridized with DNA of the specimen after pretreatment by boiling in alkaline SDS. After 4 h of hybridization the hybrids were collected onto avidin coated beads and measured. The sensitivity of the method was 1-5 x 10(5) HPV 16 DNA molecules. Cervical carcinoma cell lines CaSki and SiHa were informative as to the sensitivity of the solution hybridization and the in situ hybridization methods. CaSki cells containing about 700 HPV 16 DNA copies per cell were positive by both methods. SiHa cells with one HPV 16 DNA copy per cell were positive by the sandwich assay but remained negative in the in situ test. A series of 126 cervical scrapes collected from consecutive patients participating in a follow-up study for cervical HPV infection were tested for HPV 16 DNA by both methods. The detection rate of the sandwich test was 19/126 (15%) and that of the in situ method 21/126 (17%) yielding 26 diagnoses altogether. Twelve of these were obtained by one method only. The results obtained by studying the cervical cell lines and repeated specimens taken from constantly HPV 16 positive patients suggested that the two methods can measure different types of infections and thus complement each other in the diagnosis of cervical HPV infections.

Occurrence of Salmonella typhimurium virulence plasmid-specific sequences in different serovars of Salmonella.

We have subcloned the 96-kilobasepair (kb) virulence plasmid, pLT2, of Salmonella typhimurium line LT2 into 7 subfragments. Using these subclones as probes, 35 independent Salmonella isolates were tested for complementary DNA sequences Sequences homologous to pLT2 were present in 15 of the isolates. All of these contained sequences homologous to at least one specific probe representing 15 kb of pLT2. The traT gene from pLT2 was absent in 6 of these 15 isolates. Three strains reported to be cured of the plasmid were shown to harbour some sequences with homology to the pLT2 plasmid. Seven isolates were shown to contain homologous sequences with pBR322 but not with the pLT2 plasmid.

Screening of premalignant cervical lesions for HPV 16 DNA by sandwich and in situ hybridization techniques.

A series of 97 cervical smears and 69 directed punch biopsies derived from 84 consecutive women prospectively followed-up for cervical HPV (human papillomavirus) infections were studied using the sandwich hybridization and in situ hybridization techniques with HPV 16 DNA probes. The aim was to test the sensitivity and applicability of these two techniques in routine diagnosis of cervical HPV infections from smears. As a measure of specimen adequacy, the number of cells recovered in the cervical scrape was determined along with HPV 16 DNA in the sandwich hybridization test using human pro-alpha 2(I)-collagen gene probe. CIN (cervical intraepithelial neoplasia) was suggested in 56% of the patients by the Pap smear, and disclosed in 65% of the biopsies. HPV 16 DNA was present in 57% of cervical scrapes consistent with CIN, i.e., were of Pap smear classes III or IV. Forty percent of the scrapes not suggestive of CIN, i.e., Pap smear classes I or II, also contained HPV 16 DNA. The detection rate for HPV 16 DNA of the sandwich hybridization method was 89% of that of the in situ method in adequate scrapes, but only 43% in cell-poor specimens. The number of HPV 16 DNA-positive scrapes as compared with the total number of diagnoses obtained by studying also the biopsies was 31/36 (69 patients). The results indicate that the cervical scrape as a noninvasive specimen is applicable for screening of cervical HPV infections, and it can be studied with acceptable sensitivity by the rapid sandwich hybridization technique. However, if a punch biopsy is indicated it should be studied using the in situ hybridization technique that allows more sensitive detection of HPV DNA than any other hybridization method and enables the analysis of several HPV types in the same sample instead of only one HPV type in the scrapes.