Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation.

Comparative analysis of human papillomavirus detection by dot blot hybridisation and non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. METHODS: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. RESULTS: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). CONCLUSIONS: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.

Comparison of nucleic acid hybridization and cytologic examination for detection of human papillomavirus infection, with evaluation of two commercially available hybridization kits.

Human papillomavirus (HPV) infections were detected by analyzing exfoliated cervical cells for HPV DNA by use of nucleic acid hybridization; the results were correlated with cytologic findings on Papanicolaou smears. HPV infection was diagnosed in 154 women (20%) by either morphologic evidence on cervical smears or nucleic acid hybridization. Many of these women (38%; 58/154) exhibited Papanicolaou smears with no morphologic evidence of HPV infection. In those patients with cytologic evidence of HPV infection, only 28% were positive for HPV DNA. HPV 16 and (or) 18 were the most common types (27%) detected in women with cervical intraepithelial neoplasia, whereas all HPV groups tested were equally represented in patients with normal cervical smears. We also present an assessment of 17,000 clinical specimens submitted to this laboratory for analysis of HPV DNA.

A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1.

The sensitivity and specificity of the polymerase chain reaction (PCR) for the detection of HIV-1 proviral DNA was determined in five laboratories with extensive experience in PCR testing. Five panels consisting of 105 HIV-1-seronegative specimens from regularly repeating blood donors with no risk factors for HIV infection and 99 HIV-1-seropositive and culture-positive specimens from a cohort of homosexual/bisexual men were sent under code to each laboratory. Amplification procedures and testing algorithms by which specimens were judged positive, negative, or indeterminate varied between laboratories. The average sensitivity for the five laboratories was 99.0%, with two laboratories achieving 100%. The average specificity was 94.7%, varying between 90.5 and 100%. The overall false-positive rate was 1.8%, the false-negative rate was 0.8%, and the indeterminate rate was 1.9%. Of 1,005 determinations made by the five laboratories, 32 (3.2%) were misclassifications. Most of the classification errors occurred in specimens from uninfected individuals and were distributed among the laboratories in such a way as to indicate laboratory error rather than the inherent reactivity of some samples. This emphasizes the need for standardization of PCR testing and caution in interpreting positive PCR reactions in HIV-1-seronegative persons.

Population genetics of the highly polymorphic locus D16S7 and its use in paternity evaluation.

We have developed a DNA RFLP test to resolve paternity cases in which the accused man is included at a low probability of paternity by conventional testing. The DNA probe p79-2-23 was used to determine the allele frequency distribution for the locus D16S7 in the North American black, Caucasian, and Hispanic racial groups. Approximately 3,500 TaqI-digested DNAs were analyzed from the three populations studied. An apparent continuum of alleles was detected varying in size from 2.9 kb to 8.3 kb. Estimates of the average probability of exclusion were found to be .90 and .79 for the North American black and Caucasian populations, respectively. Gene frequency data for common and rare alleles indicated a potential paternity index ranging from 2 to 450.

Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58.

The virulence (vir) region of pTiC58 was screened for promoter activities by using gene fusions to a promoterless lux operon in the broad-host-range vector pUCD615. Active vir fragments contained the strongly acetosyringone-inducible promoters of virB, virC, virD, and virE and the weakly inducible promoters of virA and virG. Identical induction patterns were obtained with freshly sliced carrot disks, suggesting that an inducer is released after plant tissue is wounded. Optimal conditions for vir gene induction were pH 5.7 for 50 microM acetosyringone or sinapic acid. The induction of virB and virE by acetosyringone was strictly dependent on intact virA and virG loci. An increase in the copy number of virG resulted in a proportional, acetosyringone-independent increase in vir gene expression, and a further increase occurred only if an inducing compound and virA were present.

The association of the interspersed repetitive KpnI sequences with the nuclear matrix.

The KpnI sequences constitute the dominant, long, interspersed repetitive DNA families in primate genomes. These families contain related, but nonidentical sequence subsets, some of which border functional gene domains and are transcribed into RNA. To test whether these sequences perform an organizational function in the nucleus, their association with the nuclear matrix has been examined in African green monkey cells. DNase I treatment depleted the residual matrix of most of the KpnI 1.2- and 1.5-kilobase pair family sequences although significant amounts of each family remained in the loop attachment DNA fragments. Hybridization analysis of the KpnI and RsaI cleavage patterns of matrix loop attachment DNA indicate that some sequence subsets of these KpnI families are relatively less depleted than others. The nuclear matrix association of subpopulations of KpnI 1.2- and 1.5-kilobase pair families was also shown by metrizamide gradient centrifugation of nuclear matrix complexes cleaved by KpnI endonuclease. The gradients demonstrate that some KpnI segments are differentially associated with nuclear matrix proteins. Moreover, the procedures permit the preparative isolation and purification of the DNA-protein complexes containing these KpnI 1.2- and 1.5-kilobase pair sequence families. Speculations on the relationship between the matrix association of these KpnI family sequences and their possible roles in gene organization and expression are presented and discussed.