Designing HPV-specific primers for the detection of HPV-18 via multiplex PCR /
Human papillomaviruses (HPVs) were small double-stranded DNA viruses which constitute a group of more than 100 different genotypes associated with benign and malignant neoplasms of skin and epidermal tissues. It was shown that skin warts or papillomas could be transmitted between individuals by a f...
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| Format: | Book |
| Language: | English |
| Published: |
2010
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| Summary: | Human papillomaviruses (HPVs) were small double-stranded DNA viruses which constitute a group of more than 100 different genotypes associated with benign and malignant neoplasms of skin and epidermal tissues. It was shown that skin warts or papillomas could be transmitted between individuals by a filterable infectious agent. HPV types-16 and -18 were the most prevalent high-risk types of HPV which have been associated with cervical cancer throughout the world. Currently, there were several PCR-based methods for detecting HPV viruses. However, some of the commercialized kits for HPV detection and typing were too costly for screening purposes. Hypothesis was using HPV-specific primers increase the probability to detect HPV-16 and HPV-18. Objective was to design HPV-16 and HPV-18 specific primers to detect the presence of HPV DNA. Specific Objective were to design oligonucleotide primers specific for amplifying HPV types-16 and -18, and select the optimum primers using bioinformatics software, and to test the primer specificity towards HPV-16 and -18, and to evaluate the sensitivity of the PCR assay. Methods: DNA primers were designed based on E6 and E7 gene of HPV through Batch Primer 3 software and primer specification were designed through Perlprimer software. Primers would be multiplexed to enable simultaneous identification of the two HPV types. Primers were designed to create different band sizes to allow for differentiation through agarose gel electrophoresis. Optimized results would be validated by using the previously published nested PCR and DNA sequencing. Optimized method of PCR conditions would be tested on HPV DNA samples using HPV type strains purchased from American Type Culture Collection. PCR products would be analysed by gel electrophoresis separation and the PCR products would be gel-purified and subjected to automated DNA sequencing and sequence analysis. Sequence analysis would be conducted using NCBI Blastsoftware to reveal the HPV-16 and HPV-18. The sensitivity test of multiplex PCR was determined by serial dilution. Results: The primers were optimized by gradient PCR and the optimum temperature for both primers were determined as 57°C. In conclusion, the multiplex PCR using primers chosen on E6 and E7 region was proven successful to amplify and detect both HPV-16 and HPV-18 from mixedtemplate simultaneously. Thus, multiplex PCR was a valuable technique for typing HPV DNA. |
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| Physical Description: | 98 leaves : ill. ; 30cm. |