Positive Polymerase Chain Reaction Products Research Articles

Wolbachia endosymbionts in fleas (Siphonaptera).

Intracellular endosymbionts, Wolbachia spp., have been reported in many different orders of insects and in nematodes but not previously in fleas. This is the first conclusive report of Wolbachia spp. within members of the Siphonaptera. Using nested polymerase chain reaction (PCR) targeting of the 16S ribosomal RNA gene, we screened for Wolbachia spp. in fleas collected from 3 counties in Georgia and 1 in New York. The prevalence of Wolbachia spp. detected varied among the 6 different species screened: 21% in the cat flea Ctenocephalides felis (n = 604), 7% in the dog flea C. canis (n = 28), 25% in Polygenus gwyni (n = 8), 80% in Orchopeas howardi (n = 15), 94% in Pulex simulans (n = 255), and 24% in the sticktight flea Echidnophaga gallinacea (n = 101). Wolbachia spp. infection in fleas was confirmed by sequencing positive PCR products, comparing sequenced 16S ribosomal DNA (rDNA) with Wolbachia spp. sequences in GenBank using BLAST search, and subjecting sequence data to phylogenetic analysis. For further confirmation, 16S rDNA-positive samples were reamplified using the wsp gene.

Extent of digestion affects the success of amplifying human DNA from blood meals of Anopheles gambiae (Diptera: Culicidae).

The success of distinguishing blood meal sources of Anopheles gambiae Giles through deoxyribonucleic acid (DNA) profiling was investigated by polymerase chain reaction (PCR) amplification at the TC-11 and VWA human short tandem repeats (STR) loci. Blood meal size and locus had no significant effect on the success of amplifying human DNA from blood meals digested for 0, 8, 16, 24 and 32 h (P = 0.85 and 0.26 respectively). However, logistic regression found a significant negative relationship between time since ingestion and the success probability of obtaining positive PCR products among meals digested for between 8 and 32 h (P = 0.001). Approximately 80% of fresh blood meals were successfully profiled. After 8 h, the proportion of blood meals that could be successfully profiled decreased slowly with time after ingestion, dropping to below 50% after approximately 15 h. There was no significant difference in the success of amplifying human DNA from blood meals of mosquitoes killed at time 0 and 8 h after ingestion (P = 0.272).

Age-Related Mitochondrial DNA Mutations in the Human Larynx

To determine whether age-related mitochondrial DNA mutations occur in the human larynx. Genetic study of cadaveric larynx specimens. Vocal fold mucosa, thyroarytenoid muscle, and cricoarytenoidjoint tissue were harvested from 13 fresh postmortem larynges (age range, 2 d-82 y). DNA was extracted from each sample, and the polymerase chain reaction (PCR) was used to amplify a target DNA sequence resulting from the common age-associated, 4977-base-pair (bp) mitochondrial DNA deletion. PCR products were visualized by agarose gel electrophoresis. Automated sequencing determined the sequence of identified PCR products. Thirteen cadaveric larynges were obtained through the University of Kentucky Medical Center (Lexington, KY). Specimens from patients with a history of head and neck cancer, previous laryngeal trauma, or surgery were excluded. Strongly positive bands were identified in samples from three individuals. Weaker bands were seen in samples from four other samples. No band was noted from the two pediatric larynges. Different band patterns were seen among the three different tissue sites in the larynges with positive PCR products, but no consistent pattern was seen. Sequencing of the identified PCR products from selected samples confirmed that they were products of the age-associated, 4977-bp mitochondrial DNA deletion. An age-associated mitochondrial DNA deletion was detected in several post-mortem human larynges. Its presence seemed to increase in appearance with age. In the larynges in which the deletion occurred, there were individual regional differences in the occurrence of the deletion, but no consistent pattern was noted across all individuals who carried the deletion.

Detection of mycobacterial DNA in cervical granulomatous lymphadenopathy from formalin-fixed, paraffin-embedded tissue by PCR.

Cervical tuberculous lymphadenitis is the most common form of inflammatory neck mass in Korea. The diagnosis of tuberculosis requires proof of the presence of Mycobacterium tuberculosis by acid-fast staining or bacterial growth in culture. However, these are often difficult in cervical tuberculous lymphadenitis. The aim of this study was to investigate the value of the polymerase chain reaction (PCR) technique for detection of mycobacteria in routinely processed tissue sections of cervical granulomatous lymphadenopathy. In this retrospective study, twenty formalin-fixed, paraffin-embedded biopsy specimens from clinically and/or histopathologically diagnosed cervical granulomatous lymphadenopathy were analyzed for mycobacterial DNA by PCR. Two different primers to amplify mycobacterial-common 383-base pair (bp) DNA and Mycobacterium tuberculosis-complex-specific 123-bp DNA were used. Positive PCR products were sequenced directly. Mycobacterial-common DNA (383-bp positive) was found in 10 of the 20 cases. Among them, 7 cases were PCR positive with both primer sets. These seven cases can be considered as tuberculosis. The other three cases indicated possible atypical mycobacteriosis. PCR is a useful technique for the demonstration of mycobacterial DNA fragments in patients with clinically suspected cervical tuberculous lymphadenitis who have acid fast-negative histology and/or unsuccessful mycobacterial cultures.

Detection rates of TT virus among children who visited a general hospital in Japan.

Recently, genomic DNA of the novel TT virus (TTV) was isolated from patients suffering from posttransfusion hepatitis of unknown etiology. We examined sera from 197 children who visited the Department of Pediatrics at Toyohashi National Hospital. Sera were tested for TTV DNA by seminested polymerase chain reaction (PCR) using a set of primers synthesized according to the published TTV sequence. Ten children were found to be positive for TTV (5.1%). All positive PCR products were directly sequenced in both directions using a fluorescent dye terminator cycle sequencing system. The sequences were compared by a multiple sequence alignment and a phylogenetic tree was constructed. The phylogenetic tree showed that two of the TTV isolates found in the present experiment did not belong to any of the phylogenetic groups previously reported.

Molecular evaluation of enteroviruses in the pathogenesis of idiopathic dilated cardiomyopathy.

There is great disparity in the literature as to the presence and relevance of enterovirus in heart tissue from patients with dilated cardiomyopathy (DCM). Published estimates of enteroviral positive tissue in DCM range from 0 to 50%. Very little sequence information is so far available on those samples which are positive. Re-examination of fresh biopsy material from patients previously tested, plus 13 new cases of DCM, and sequencing the products would yield information on the validity of the technique and on the type of virus being detected. RNA from biopsy or explant tissue was tested for the presence of enterovirus using reverse transcriptase polymerase chain reaction (PCR). The nucleotide sequences of all positive PCR products were determined by direct sequencing. Positive PCR signals were found in 10% of samples from patients with DCM and in 16% of control tissues. Two DCM and 12 control samples gave the same nucleotide sequence, which was different from the CB3 used as a positive control. The other 4 DCM samples all produced multiple bands on sequencing. The results do not support a major role for enterovirus in DCM. There is need for some caution, however, as a review of the literature shows that studies using single biopsies, such as this one, produce consistently lower estimates for enterovirus than do those wherein multiple biopsies are examined.

Latent adenoviral infection in follicular bronchiectasis.

The follicular form of bronchiectasis originally described by Whitwell (1) has been associated with adenoviral infection. The present study compares resected lungs from 16 patients with follicular bronchiectasis (in 10 of whom a nonviral etiology was identified) with those from eight patients with a nonfollicular histologic pattern. DNA isolated from sections of 45 paraffin-embedded lung samples was subjected to the polymerase chain reaction (PCR) using primers for the E1A region of the adenovirus genome and the human HLA DQ alpha gene. In situ hybridization (ISH) was performed on separate sections cut from the same blocks using a probe for the entire adenovirus 5 genome. E1A was demonstrated in six of eight patients (75%) with non-follicular bronchiectasis (non-FB) and in four of 16 (25%) with follicular bronchiectasis (FB) (p < 0.03, Fisher's exact test). The optical density ratio for the E1A product of PCR (ratio of E1A product from specimen to that from 1 pg adenovirus DNA) was significantly lower in FB than in non-FB (0.057 +/- 0.054 versus 0.365 +/- 0.223 [mean +/- SEM], p < 0.05). Moreover, the duration of symptoms of bronchiectasis in patients without E1A in bronchial specimens was significantly shorter than that of patients with positive E1A PCR products (3.09 +/- 1.44 versus 14.41 +/- 3.33 yr; p < 0.05). By ISH, adenovirus was demonstrated in three patients with FB and in two with non-FB (17.2 and 18.8% of tissue blocks, respectively; NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerase chain reaction for diagnosis of porcine proliferative enteropathy

A polymerase chain reaction (PCR) assay for detection of the intracellular bacteria, ileal symbiont intracellularis of porcine proliferative enteropathy is described. The testis based on specific DNA primers and gave positive PCR product from samples of preserved intestinal mucosa and faeces from affected pigs. Mucosa and faeces from normal pigs gave no positive PCR products. The identity of the PCR product was confirmed by DNA-DNA hybridization with a probe, pCLO78, specific for IS intracellularis. Positive results were only observed in animals with active lesions of proliferative enteropathy. PCR is probably the most useful method for diagnosis of prolifrative enteropathy that is currently available for live animals.

Varicella‐zoster virus DNA in recurrent aphthous ulcers

The present study was undertaken to examine for varicella-zoster virus (VZV) constituents in recurrent aphthous ulcers (RAU). Smears and biopsies of ulcers 0-3 days old were obtained from 20 otherwise healthy minor/major RAU patients and from two patients with Behçet's syndrome. VZV could not be cultivated from any of the biopsies, all smears were negative for VZV antigen, and in situ hybridization on the five samples tested was negative for VZV nucleic acid. DNA extracted from biopsies, however, yielded positive polymerase chain reaction (PCR) products in all cases with one primer set of the protein kinase 66 (PK66). Ten control biopsies from non-RAU subjects yielded no PCR products apart from the sample from AP, who obtained the biopsies, and from HOM, who performed the PCR, suggesting that the DNA had been passed on from the patients. Other parts of the PK66 gene and of thymidine kinase 36 yielded no products. Although the consistent presence of VZV DNA in RAU might suggest a pathogenetic implication, the potential presence and possible pathogenetic importance of other herpesviruses cannot be excluded.