We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high-and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.Accumulating evidence indicates that persistent infection with high-risk human papillomaviruses (HPVs) is indeed a major causative factor in the development of cervical intraepithelial neoplasia and invasive cervical carcinoma (42,10,8,5,40,27,11,30). The HPV family includes over 100 genotypes, 30 to 40 of which are mucosotropic, and at least 15 types of the mucosotropic HPVs have been linked to cervical cancer (5,8,10,42). In addition, some of these types are also related to other cancers of the genital tract (21,22) and to cancers of other organs (14, 28). Light microscopic examination of a Papanicolaou (Pap)-stained smear is of primary importance for the detection of cervical cancer precursor lesions. It has been demonstrated that concomitant testing for DNA of the highrisk HPV types by the Pap test can clearly identify women at high risk for cervical cancer, particularly if persistent infection by high-risk HPV types is diagnosed (5,8,10,11,27,30,40). Furthermore, HPV genotyping is of critical importance for the investigation of the clinical behavior and the epidemiology of HPV infection, for population studies for HPV vaccination trials, and for monitoring of the efficacy of HPV vaccines. Several genotyping methods have been developed in order to identify high-risk HPV in liquid-based cytology (LBC) samples and tissue samples (1,12,34). The molecular techniques that have been applied for HPV DNA detect...

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 x 10(-4) M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis.

Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC ¼ 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors. Cancer Prev Res; 4(5); 674-83. Ó2011 AACR.