Consensus Polymerase Chain Reaction Assay Research Articles

Helicobacter pylori: Detection of iceA1 and iceA2 Genes in the Same Strain in Mexican Isolates

Helicobacter pylori iceA1 and iceA2 gene amplification is usually performed to identify mixed populations as both genes are apparently reportedly exclusive. However, some strains isolated from Mexico show both iceA genes. The aim of this study was to establish the frequency of these genes in Mexican isolates and genomic diversity of the H. pylori strains. One hundred thirty six biopsies were obtained from 68 patients (39 children and 29 adults). The presence of H. pylori was confirmed in 3/18 children and 6/19 adults by culture. There were 93 clinical strains isolated from nine patients. Additionally, we studied 37 strains from a strain collection isolated from 10 patients. The strains were genotyped and dual iceA genes were identified by polymerase chain reaction (PCR) and amplicons were sequenced. In addition, an enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assay was performed as fingerprinting method. The genotypification of the H. pylori isolates indicated that all strains were vacA+; 86% babA2+, 86% cagA+, 82% vacA s1m1+, 19% iceA1+, 9% iceA2+, and 72% of them carried both iceA1 and iceA2 genes. The ERIC-PCR profiling revealed that the strains clustered in eight genetic groups depending on the presence of iceA1, iceA2 or both. A basic local multiple alignment analysis of the nucleotide sequences revealed that the iceA1 and iceA2 genes exhibited no relevant similarity. The results here showed the presence of triple-positive strains (babA, cagA, vacA) of H. pylori and strains carrying simultaneously both iceA1 and iceA2 genes.

Association between simian virus 40 DNA and lymphoma in the United kingdom.

Recent studies have reported the presence of simian virus 40 (SV40) DNA sequences in approximately 40% of tumor samples from non-Hodgkin's lymphoma (NHL) patients from the United States. We examined a series of 259 tumor and blood samples, including 152 NHL samples, from patients in the U.K. with lymphadenopathy and lymphoid leukemia for the presence of SV40 DNA using a highly sensitive quantitative polymerase chain reaction (PCR) assay and a consensus PCR assay capable of detecting the polyomaviruses SV40, BK, and JC. SV40 DNA sequences were not detected in any sample using either assay. Because the incidence of NHL is similar in the U.K. and the United States, this finding suggests that SV40 is unlikely to have an etiologic role in NHL.

Approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs.

The identification of porcine viruses so far unrecognized is required to minimize virus-related risks associated with xenotransplantation. We used a pan-herpes consensus polymerase chain reaction assay to search for unrecognized porcine species of the Herpesviridae. The assay targets conserved regions of the herpesvirus DNA polymerase (DPOL) gene, using primers that were modified to diminish the assay's recognition capacity for the highly prevalent porcine lymphotropic herpesviruses 1, 2 and 3 (PLHV-1, -2, -3), without substantially lowering the universal detection capacity of the assay. Analysis of 495 porcine blood and tissue samples from 294 animals, including 35 samples from 20 immunosuppressed pigs, resulted in the amplification of 128 herpesviral DPOL sequences. Sequence analysis attributed 127 of the amplimers to the known porcine herpesviruses (PLHV-1, -2, -3; porcine cytomegalovirus; pseudorabiesvirus). In none of the pig samples analyzed here, evidence was obtained for the presence of additional novel porcine herpesvirus species. Therefore we conclude that pigs bred for the purpose of xenotransplantation pose a negligible risk of transmitting presently unrecognized herpesviruses to organ recipients.

Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses—infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)—in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583–590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.

Risk factors for oral human papillomavirus in adults infected and not infected with human immunodeficiency virus.

To investigate in a cross-sectional study the determinants of oral human papillomavirus infection in 287 individuals who are sexually active. To assess prevalence as well as risk factors for oral human papillomavirus infection. One hundred seventy-eight human immunodeficiency virus-seropositive (158 men and 20 women) and 109 human immunodeficiency virus-negative (73 men and 36 women) individuals were recruited consecutively from sexually transmitted disease-human immunodeficiency virus clinics and gastrointestinal endoscopy clinics. Oral brushings were tested with the L1 consensus polymerase chain reaction assay for human papillomavirus detection. Human papillomavirus DNA was detected in 32 (11.2%) of 287 individuals. Associated with oral human papillomavirus infection on univariate analyses were human immunodeficiency virus infection (odds ratio, 6.9; 95% confidence interval, 2.0-23.2), homosexuality (odds ratio, 3.7; 95% confidence interval, 1.5-9.4), unprotected oral sex (odds ratio, 5.5; 95% confidence interval, 1.6-18.4), syphilis (odds ratio, 2.5; 95% confidence interval, 1.1-6.3), gonorrhea (odds ratio, 4.2; 95% confidence interval, 1.9-9.1), Chlamydia trachomatis (odds ratio, 4.4; 95% confidence interval, 1.8-10.6), and genital herpes (odds ratio, 2.9; 95% confidence interval, 1.3-6.5). Human immunodeficiency virus infection and C. trachomatis were independently predictive of human papillomavirus infection in multivariate stepwise logistic regression.