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M Ranki

Publications and source records attributed to M Ranki.

At least 19 recordsLinked to original sources
PubMed1992

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.

Adolescent↗
PubMed1992

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.

Animals↗
PubMed1992

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.

DNA, Viral↗
PubMed1992

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.

Base Sequence↗
PubMed1991

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.

Bacterial Infections↗
PubMed1990

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.

Cervix Uteri↗
PubMed1990

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.

Base Sequence↗
PubMed1990

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.

Cervix Uteri↗
PubMed1989

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.

Cloning, Molecular↗
PubMed1989

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.

Cell Line↗
PubMed1988

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.

DNA, Viral↗
PubMed1986

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.

2-Acetylaminofluorene↗
PubMed1986

Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping.

Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay.

Cervix Uteri↗
PubMed1986

Serological evidence for the role of bacterial infections in the pathogenesis of thyroid diseases.

Subacute thyroiditis is generally believed to be of viral origin, and infection is also suspected of playing a role as a triggering factor in the pathogenesis of autoimmune thyroid diseases. We have measured a broad spectrum of bacterial and viral antibodies in paired sera of 32 patients with thyroid disease of recent onset. The data indicate a preceding infection in 14 (44%) of the patients, enterobacterial in 5, streptococcal in 4 and staphylococcal in 2. A viral infection was suggested in 6 patients, in each case caused by different agents; 3 of them also showed evidence of a bacterial infection. Patients with positive microbial serology were found in all diagnostic groups, including subacute thyroiditis, Graves' disease and Hashimoto's disease. These results suggest an association between a preceding bacterial infection and the development of thyroid disease in some patients.

Adult↗
PubMed1986

Rapid diagnosis of respiratory adenovirus infections in young adult men.

Rapid viral diagnosis was attempted in 106 military conscripts with pneumonia and in 101 military conscripts with other types of respiratory infections. Nasopharyngeal suction specimens (NPS) were assayed for viral antigens by immunofluorescence and enzyme immunoassay (EIA). Sputum specimens from 97 pneumonia patients were assayed for viral antigens by EIA. Also, 71 NPS and 13 sputum specimens were examined for the presence of adenovirus DNA by a sandwich hybridization (HYB) method. The reference test was adenovirus isolation in cell culture from the NPS. Adenoviruses were isolated from 6 pneumonia patients and from 20 patients with other respiratory infections. Of these 26 NPS, rapid diagnosis was successful in 13, 16, and 14 cases by EIA, immunofluorescence, and HYB, respectively. Four antigen-positive specimens were found among the 181 specimens which were negative by virus isolation. Sputum was found to contain adenovirus antigen by EIA in 5 of 97 tested specimens. Of these 97 specimens, 13 were selectively tested in HYB, and a positive signal was observed in 4 cases. Serological testing of paired sera revealed 23 adenovirus infections in the pneumonia group and 42 in the group with other respiratory infections. Other viral infections were found only sporadically. All rapid virus detection methods showed excellent specificity but had a lower sensitivity (60%) than virus isolation. Our results show that rapid methods for diagnosing respiratory adenovirus infections can be successfully used in selected groups of adults.

Adenoviridae Infections↗