An-Najah Staff

Paratuberculosis is an endemic disease and induces high economical losses in Jordan. There is no information available on genotypic variation of Mycobacterium avium subsp. paratuberculosis (MAP) isolated from animals in Jordan. In this study, we investigated 150 fecal samples from sheep, goats, and cattle for the presence of paratuberculosis using bacterial culture and polymerase chain reaction (PCR)-RFLP analysis of insertion sequence IS1311. Analysis of the results revealed that genotypic information from sheep, goat, and cattle could classify them into cattle or sheep strains. All culture isolates from cattle, 12.5% of the isolates from sheep, and 50% of the isolates from goats were cattle strain, while 87.5% of the isolates from sheep and 50% of the isolates from goats were sheep strain. Sequencing of the IS1311 268 bp PCR product from the three animal species confirmed the different MAP patterns.

We established a new assay to detect the E6–E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.