Polymerase Chain Reaction Primer Sets Research Articles

Genome size and microsatellites: the effect of nuclear size on amplification potential.

Although the frequency of microsatellite DNA regions generally increases with increasing genome size, genome size has a negative effect on polymerase chain reaction (PCR) amplification. Thus, researchers developing sets of PCR primers, as is commonly done for microsatellite DNA regions, may encounter greater difficulty when working with species that have larger genomes. I investigated the effect of genome size on overall amplification success using data from nine different metazoan taxa. The proportion of primer sets that did not amplify PCR products was strongly and positively correlated with the haploid C value of the target species. Increasing genome size may affect amplification success negatively because of a decrease in target:nontarget DNA or by dilution of the available primer pool by nonspecific binding.

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.

To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) Either single round or second round (seminested) PCRs were developed and validated. Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% ( n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1–3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

Detection of Human Papillomaviruses in Cervical Neoplasias Using Multiple Sets of Generic Polymerase Chain Reaction Primers

Objective.The aim of this study was to evaluate precisely the differences in the spectra of human papillomavirus (HPV) types detected by different generic primer pairs commonly used for detection of this extraordinarily heterogeneous virus.Methods. Three sets of polymerase chain reaction (PCR) primers for the L1 open reading frame (ORF) and two sets for E6/E7 ORFs were used to detect HPVs in DNAs from 107 cervical tissues, including 77 cervical neoplasias. HPV types were determined by analysis of restriction fragment length polymorphisms (RFLPs) and nucleotide sequencing.Results. A high overall detection rate of HPV in cervical neoplasias (76/77, 98.7%) was achieved by polymerase chain reaction (PCR) amplification with multiple sets of generic primers, while the detection rate for each individual primer pair varied from 48/77 (62%) to 70/77 (91%). Only in 34 of 77 cases (44%) were HPV DNAs positive for all sets of primer pairs. Further determination of HPV types by RFLPs and nucleotide sequencing showed inconsistencies between the PCR primer pairs used.Conclusion. Our study revealed that the HPV detection rate is critically affected by the choice of PCR primers, and that appropriate use of combinations of generic PCR primer sets followed by RFLP analyses is both necessary and sufficient for typing most HPVs in cervical lesions. More precise methods such as sequencing would be necessary in only a few cases.

Direct polymerase chain reaction from whole blood without DNA isolation.

Blood and other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), so that isolation of DNA is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances and makes DNA isolation from blood unnecessary for PCR. When this reagent was included in the PCR mixture, DNA fragments of the beta-globin gene could be efficiently amplified directly from human blood samples treated with various anticoagulants or PCR-inhibitory substances. We confirmed the usefulness of this cocktail by examining a large number of blood samples with various PCR primer sets. In addition to fresh blood, this method enabled PCR amplification from blood samples stored at 4 degrees C, -20 degrees C or -80 degrees C for a minimum of 1 year.

Development of PCR‐Based Markers for a High Grain Protein Content Gene from Triticum turgidum ssp. dicoccoides Transferred to Bread Wheat

Grain Protein Content (GPC) of wheat (Triticum aestivum L. and T. turgidum L.) is important for improved nutritional value and is also one of the major factors affecting breadmaking and pasta quality. A quantitative trait locus (QTL) for high GPC was detected a few years ago in the short arm of chromosome 6B from accession FA15‐3 of Triticum turgidum L. var. dicoccoides New molecular markers are presented here to facilitate the transfer of this high GPC gene into tetraploid and hexaploid wheat cultivars. Two sets of PCR (polymerase chain reaction) primers were designed to amplify regions of the non‐transcribed spacer of the XNor‐B2 locus. This locus was selected because it mapped on the peak of the QTL for GPC. The first pair of allele‐specific primers produced an amplification product only when the T. turgidum var. dicoccoides XNor‐B2 allele was present. The second pair of primers amplified fragment(s) of similar length in the different genotypes that after digestion with the restriction enzyme BamHI allowed differentiation of the T. turgidum var. dicoccoides allele. Four microsatellites markers were mapped on the short arm of chromosome 6B at both sides of the QTL peak and two on the long arm. Five additional amplified fragment length polymorphism (AFLP) markers were mapped into the QTL region on 6BS. These PCR markers together with 10 restriction fragment length polymorphism (RFLP) markers showed that the hexaploid cultivar Glupro, selected for high GPC, carries a distal segment of chromosome 6BL and a proximal segment of 6BS from dicoccoides accession FA15‐3 encompassing the segment with highest LOD score for the GPC QTL.

No Polymorphism in the Tissue Transglutaminase Gene Detected in Coeliac Disease Patients

Background: The autoantigen for the anti-endomysial antibody (AEA) found in coeliac disease has recently been reported to be the enzyme tissue transglutaminase (tTG). Polymorphisms in the gene for tTG would result in different enzymic isoforms being expressed. Certain isoforms may interact with gliadin to create antigenic neoepitopes, which could then generate an immune response in genetically predisposed individuals possessing major histocompatibility complex (MHC) class II DQ2. Methods: We have sequenced the coding region of tTG in coeliac patients and normal controls. Total RNA was extracted from mucosal biopsies from eight AEA-positive histologically proven coeliac disease patients and four control patients with a histologically normal duodenum and a negative AEA. The 2-kb coding region of tTG was amplified in overlapping fragments by reverse transcription polymerase chain reaction (PCR), using five sets of PCR primers. Each overlapping PCR fragment was sequenced using the fmol DNA sequencing system. Results: tTG transcripts were detected in all samples. There was no difference in the coding sequence of tTG between the four control and eight coeliac patients, even though we observed differences in sequence between our study and the original published sequence. These differences have also been reported in sequences published subsequent to the original description. Conclusions: Polymorphisms in the tTG gene have not been observed in coeliac disease patients and therefore cannot explain the creation of neoepitopes.

Species‐specific polymerase chain reaction primers for Lactococcus garvieae

A dihydropteroate synthase gene from the chromosomal DNA of the fish‐pathogenic bacteria Lactococcus garvieae (formerly Enterococcus seriolicida) was cloned. This gene was then chosen as the target for polymerase chain reaction (PCR). The designated PCR primer set only amplified a 709‐bp DNA fragment from L. garvieae strains, and did not amplify the same molecular size fragment from related species of L. lactis, Enterococcus faecalis, E. faecium or β‐haemolytic Streptococcus sp. The kidney tissue of yellowtail, Seriola quinqueradiata (Temminck and Schlegel), a species which is naturally infected with L. garvieae, and also kidney tissue samples of healthy yellowtail were stored in TNES‐Urea. The DNA was extracted from tissue samples by a modification of the standard method and by a boiled‐extraction method. In particular, template DNA was utilized within 30 min following extraction and purification by the boiled‐extraction method. These species‐specific PCR primers could amplify a L. garvieae target sequence from yellowtail which were naturally infected with L. garvieae. The total procedure for the diagnosis of L. garvieae infections in fish, from the point of DNA extraction to observation in an agarose gel following electrophoresis, can be performed in less than 4 h.

Isolation by PCR-based methods of a plant antifungal polygalacturonase-inhibiting protein gene

A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple fruit was cloned by degenerate oligo-primed polymerase chain reaction (PCR) and Inverse PCR. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design Inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for Inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. A new set of PCR primers were designed to the 5' and 3' ends of the gene, which allowed amplification of the fulllength gene from apple genomic DNA. This method has broad application to isolation of homologues of any gene for which some sequence information is known.

Polymerase Chain Reaction (PCR)-based Typing Analysis of Atypical Isolates of the Fish Pathogen Aeromonas salmonicida

Two hundred and five isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and countries were characterized by polymerase chain reaction (PCR) targeting four genes. The chosen genes were those encoding the extracellular A-layer protein (AP), the serine protease (Sprot), the glycerophospholipid:cholestrol acetyltransferase protein (GCAT), and the 16S rRNA (16S rDNA). All the atypical A. salmonicida isolates could be assigned to 4 PCR groups. Group 1 comprised 45 strains which tested positive for PCR amplification, using the 16S rDNA, GCAT2, Sprot2, and AP primer-sets. Group 2 comprised 88 strains with produced PCR products using the 16S rDNA, GCAT2 and AP primer-sets. Group 3 comprised 21 strains which produced PCR products using 16S rDNA, GCAT2 and Sprot2 primer-sets, and group 4 comprised 51 strains which produced PCR products using the 16S rDNA and GCAT2 primer-sets only. A. salmonicida subsp. salmonicida isolates tested, belonged to group 1. The PCR primer-sets separated A. salmonicida from other reference strains of Aeromonas species and related bacteria with the exception of Aeromonas hydrophila. The results indicated that PCR typing is a useful framework for characterization of the increasing number of isolations of atypical A. salmonicida.

Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction employing patients' sputum.

A preliminary evaluation of the diagnostic potential of a polymerase chain reaction (PCR) assay using diurnally collected sputum from bancroftian filariasis patients is described. A new set of PCR primers amplifying a 254-bp-long sequence termed AccI, derived from a long dispersed repeated sequence and SspI primers previously employed for PCR-based diagnosis were employed in this study with similar results. Of the 34 sputum samples from patients, 32 (94%) were PCR positive. Of the 18 patients with low to high microfilaremia (21-1560 microfilariae/ml), 16 (88.8%) were PCR positive. Of the remaining 16 patients, 6 with very low microfilaremia (2-6 microfilariae/ml) and 10 without microfilaremia, all (100%) were PCR positive. Two PCR-positive cases among the 13 endemic normal individuals tested (15.4%) may represent cases of occult filariasis. PCR amplification was also demonstrated with one PCR-positive sputum aliquot when mixed with 14 sputum aliquots from uninfected (PCR-negative) individuals. The potential diagnostic merits of the sputum-PCR assay are discussed.

Nucleic Acid Persistence in Heat-Killed Escherichia coli O157:H7 from Contaminated Skim Milk

Polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR using primers targeting 16S rRNA sequences in Escherichia coli O157:H7 were applied to monitor the stability of rDNA and rRNA in cells killed by mild heat treatment (60°C) in skim milk. Serial dilutions of purified RNA and DNA from E. coli 0157:H7 in skim milk were amplified by RT-PCR or PCR, respectively, before heat treatment and at time points 0, 6, 12, 24, and 48 h after heating. In general, DNA-PCR provided stronger amplification signals compared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a lower efficiency of RNA amplification compared to that of DNA. Ribosomal RNA and rDNA could be amplified by RT-PCR or PCR from both viable and dead cells throughout the 48-h posttreatment holding period. For RT-PCR, amplification signals decreased in intensity with increased holding time, while the efficiency of amplification of DNA sequences from dead cells remained fairly stable throughout the study. DNA persistence was greater than that of rRNA following cell death by mild heat treatment in skim milk. Skim milk did not appear to accelerate nucleic acid degradation. While rRNA was less stable than DNA, its detection by RT-PCR may not be appropriate as an exclusive indicator of cell viability in minimally processed foods.

Detection ofNeisseria Gonorrhoeae by polymerase chain reaction (PCR)

Three different sets of primers were designed using FASTA homology search and PRIMERSELECT for the specific detection ofNeisseria gonorrhoeae using polymerase chain reaction (PCR). These primers amplified the highly conserved regions of genes for Open Reading Frame (ORF), Outer Membrane Protein (OMP) and 23S rRNA sequences ofN. gonorrhoeae. Each of the PCR primer set was evaluated using the DNA samples isolated from eight different positive isolates ofN. gonorrhoeae cultured from urethral swabs of patients visiting Maulana Azad Medical College and Safdarjung Hospital. Amplification products were analyzed on agarose gel electrophoresis. Two sets of PCR primers, designated as Ngu1/Ngu2 and Ngu5/Ngu6, specific for ORF and OMP gene respectively, amplified four regions of the gene which may help to differentiate the various strains ofN. gonorrhoeae infecting indigenous population. In contrast, a single, specific PCR product of 650 bp was visualized on agarose gel with primers Ngu3/Ngu4, amplifying the 23S rRNA gene. Under optimum conditions, as low as 25ng of DNA isolated from eight different clinical strains ofN. gonorrhoeae could be detected by PCR using Ngu3/Ngu4 set of primers. Our results suggested that Ngu3/Ngu4 could serve as good primers for the specific, reproducible and sensitive diagnosis ofNeisseria gonorrhoeae from clinical samples.

Highly Variable Microsatellites in the California Market Squid Loligo opalescens.

: Attempts to study the genetic population structure of cephalopods are impeded by the low levels of genetic variation in these species. We have developed polymerase chain reaction (PCR) primers for six hypervariable microsatellite markers in order to analyze the molecular population structure in the Californian market squid Loligo opalescens. Each of these genomic loci has been cloned and fully sequenced. Here we report the sequence and properties of the six PCR primer sets for the amplification of hypervariable microsatellites. Heterozygosity levels in six squid samples from different locations are high for all loci tested.

Enteroviral genome in native hearts may influence outcome of patients who undergo cardiac transplantation.

The Enterovirus may be the most common agent responsible for viral myocarditis and cardiomyopathy. Very little of the literature is available concerning the follow-up of patients who underwent transplantation with enteroviral positivity in native hearts. In the present study, 45 explanted hearts from patients who underwent orthotopic heart transplant at University of Padova were studied by reverse transcriptase (RT)-polymerase chain reaction (PCR): 27 patients had dilated cardiomyopathy (DC), 12 had ischemic cardiopathy (IC), 2 had valvular disease (VD), 2 had arrhythmogenic right ventricular cardiomyopathy (ARVC), 1 had giant cell myocarditis (GCM), and 1 had lymphocytic myocarditis (LM). Two sets of PCR primers from the highly conserved region of Enterovirus and Rhinovirus were used. Samples of both ventricles and septum were analyzed in every patients. The RT-PCR and nucleotide sequencing of amplicons were also performed on all post-transplantation follow-up biopsies in patients with Enterovirus positivity in the native heart. The viral genome was detectable in only 1 of 27 patients with DC (3%) and in 1 patient with LM. Nucleotide sequence analysis of the amplified product showed differences in nucleotide sequence of PCR samples compared with the sequence of the coxsackievirus B3 used in the current study. The patient with Enterovirus-positive DC showed a higher index of severe rejection (>3A) in the first 6 months, compared with the other patients tested. The patient with Enterovirus-positive LM died of disease recurrence 2 months after transplantation. The present study reveals a scarce presence of Enterovirus in the myocardium of patients with chronic myocardial disease. Because Enterovirus infection was predictive of a poor prognosis in these two patients, molecular studies are useful in excluding viral involvement in native hearts of transplanted patients.

Development of a PCR test for rapid diagnosis of contagious equine metritis.

In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of experimentally infected mares with sensitivity comparable to that of bacterial isolation. Furthermore, two-step PCR was more sensitive than the culture method. Upon examination of field samples, 12 out of 3,123 samples were positive by single-step PCR while only 2 were positive by bacterial culture. The 12 PCR-positive samples originated from 5 mares, of which 3 animals were considered to be carriers based on previous bacteriologic and serologic diagnoses for CEM. The PCR test described in this study would provide a specific and highly sensitive tool for the rapid diagnosis of CEM.

The value of microsatellite instability in the detection of HNPCC families and of sporadic colorectal cancers with special biological features: An investigation on a series of 100 consecutive cases

Microsatellite instability (MI) is a biological characteristic of most tumors involved in hereditary non-polyposis colorectal cancer (HNPCC). This disease appears to be caused by germline mutations in mismatch repair (MMR) genes, which are responsible for repairing single base-pair mismatches. At least five human genes participate in MMR. MI also occurs in 10%-15% of sporadic colorectal cancers. Because MI detection has been suggested as an alternative diagnostic tool for identification of HNPCC families, in this study we analyzed the MI pattern in 100 consecutive colorectal carcinomas in order to correlate them with the clinicopathologic features and family histories of the patients. A series of 100 colorectal cancers was evaluated for MI with 10 polymerase chain reaction primer sets. Instability results were compared with family history and other clinical and biological characteristics. MI was detected in 36 of 100 cancers, 27 of which showed low instability and nine a high instability. The low- and high-instability cases showed similar clinicopathological characteristics, and significantly positive associations were observed between MI and mucinous histological type (P = 0.0001) and MI and peritumoral lymphocytic infiltration (P = 0.01). A single HNPCC family was identified in the high-grade MI group, while two families belonged to the MI-negative group. Our data suggest that MI screening is probably not an efficient strategy for identifying HNPCC cases. MI does, however, appear capable of defining a category of colorectal cancers with favourable prognostic features and should be investigated at least in all cases of mucinous adenocarcinomas.

Human Papillomaviruses are Commonly Found in Normal Skin of Immunocompetent Hosts

We have previously demonstrated, by the combined application of two degenerate polymerase chain reaction primer sets, the presence of human papillomavirus (HPV) DNA in 91% of cutaneous squamous cell cancers from renal allograft recipients, with multiple types being present in one-third of these tumors. Five HPV types--HPV 20, HPV 23, HPV 38, DL40, and DL267--accounted for 73% of positive results. These HPV types are all related to the epidermodysplasia verruciformis group, and HPV 38 was originally isolated from a melanoma. The aims of this study were to determine: (i) whether HPV DNA could readily be demonstrated in skin tumors, as well as in perilesional skin, of immunocompetent patients using two polymerase chain reaction primer sets; (ii) the prevalence of infections in normal skin; and (iii) the prevalence of HPV 38 or HPV 38 related viruses in melanoma. The HPV types detected in lesions from renal allograft recipient were present not only in the perilesional skin and tumors of immunocompetent patients, but also in 35% of normal skin biopsies. HPV DNA was present in 13% of the melanoma samples, but none harbored HPV 38 DNA. We identified four putatively new HPV types. Infections with different types of human papillomavirus are widespread and often occur in clinically normal skin. In vitro studies are required to determine the specific molecular mechanisms by which these HPV types may be involved in the etiology of nonmelanoma skin cancer.

A multifaceted study of human papillomavirus and prostate carcinoma

The presence of human papillomavirus (HPV) in the prostate and its role in prostate carcinoma are in dispute. To address these issues, two laboratories with extensive HPV experience were selected to test specimens from two populations at different risk for prostate carcinoma, using three different polymerase chain reaction (PCR) assays and two serologic assays for HPV. The cases were comprised of 51 African-American (men at high risk for prostate carcinoma) and 15 Italian (men at intermediate risk for prostate carcinoma) men with prostate carcinoma. Controls were 108 African-American men and 40 Italian men with histologically proven benign prostate hypertrophy (BPH). Prostate tissue was obtained from each patient at surgery and immediately frozen in liquid nitrogen. The PCR primer sets included two (MY09/MY11 and GP5+/ GP6+) that amplify different regions of L1 and a third (WD66,67,154/WD72,76) targeted to E6. Sensitivity in the 2 L1 PCR assays was shown to be 1 HPV DNA genome per 100 cells. Serum antibodies to HPV-16 and HPV-11 virus-like particles (VLPs) were detected using enzyme-linked immunosorbent assays. All available prostate carcinoma tissue specimens (n = 63) and BPH specimens from selected controls (n = 61) were tested by PCR. Human beta-globin DNA could be amplified from all specimens except three carcinomas, but no HPV DNA was detected in any case or control specimens by MY09/MY11 or E6 PCR. Microdissection of 27 carcinoma specimens was conducted to minimize nontumor DNA, but results remained negative by MY09/MY11 and GP5+/GP6+ PCR. In addition, serum specimens in cases (n = 63) and controls (n = 144) showed no differences in their responses against HPV-16 (P = 0.54) or HPV-11 VLPs (P = 0.64). The findings suggest that HPV is not associated with prostate carcinoma, and that HPV DNA is not at all common in the prostate glands of older men.

A multifaceted study of human papillomavirus and prostate carcinoma

BACKGROUND The presence of human papillomavirus (HPV) in the prostate and its role in prostate carcinoma are in dispute. To address these issues, two laboratories with extensive HPV experience were selected to test specimens from two populations at different risk for prostate carcinoma, using three different polymerase chain reaction (PCR) assays and two serologic assays for HPV. METHODS The cases were comprised of 51 African-American (men at high risk for prostate carcinoma) and 15 Italian (men at intermediate risk for prostate carcinoma) men with prostate carcinoma. Controls were 108 African-American men and 40 Italian men with histologically proven benign prostate hypertrophy (BPH). Prostate tissue was obtained from each patient at surgery and immediately frozen in liquid nitrogen. The PCR primer sets included two (MY09/MY11 and GP5+/GP6+) that amplify different regions of L1 and a third (WD66,67,154/WD72,76) targeted to E6. Sensitivity in the 2 L1 PCR assays was shown to be 1 HPV DNA genome per 100 cells. Serum antibodies to HPV-16 and HPV-11 virus-like particles (VLPs) were detected using enzyme-linked immunosorbent assays. RESULTS All available prostate carcinoma tissue specimens (n = 63) and BPH specimens from selected controls (n = 61) were tested by PCR. Human β-globin DNA could be amplified from all specimens except three carcinomas, but no HPV DNA was detected in any case or control specimens by MY09/MY11 or E6 PCR. Microdissection of 27 carcinoma specimens was conducted to minimize nontumor DNA, but results remained negative by MY09/MY11 and GP5+/GP6+ PCR. In addition, serum specimens in cases (n = 63) and controls (n = 144) showed no differences in their responses against HPV-16 (P = 0.54) or HPV-11 VLPs (P = 0.64). CONCLUSIONS The findings suggest that HPV is not associated with prostate carcinoma, and that HPV DNA is not at all common in the prostate glands of older men. Cancer 1998;82:1118-25. © 1998 American Cancer Society.