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Title: Molecular diagnostics of human papillomavirus by genotyping DNA arrays
Other Titles: Li yong ji yin fen xing xin pian ji shu dui ren lei ru tou liu bing du jin xing fen zi zhen duan
Authors: Ji, Shenglin (季晟琳)
Department: Dept. of Biology and Chemistry
Degree: Master of Philosophy
Issue Date: 2007
Publisher: City University of Hong Kong
Subjects: DNA microarrays -- Diagnostic use
Molecular diagnosis
Papillomavirus diseases -- Molecular diagnosis
Notes: CityU Call Number: RB43.8.D62 J5 2007
Includes bibliographical references (leaves 91-101)
Thesis (M.Phil.)--City University of Hong Kong, 2007
xiv, 116 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: High throughput screening for infectious pathogens is an important aspect in clinical diagnostics. Cervical cancer is the second biggest cause of women cancer mortality. Human papillomavirus (HPV) infection is the single main risk factor for the development of cervical cancer. There are increasing demands using HPV diagnosis tests for screening of cervical cancer or its precursors. In this project, a system was constructed targeting miniaturization, low cost and high throughput screening of HPV using membrane-based genotyping DNA arrays. The principle of this platform is straightforward, and the diagnostic process is simple and fast. 5’ amino-modified type-specific oligonucleotide probes are immobilized on nylon membranes through chemical reactions and arranged in an array format, which are small enough to fit into 48-well plate or even 96-well plate. PCR targeting HPV L1 region is carried out by optimized biotinylated consensus primers. The end-labeled PCR products hybridize to the immobilized probes on membranes, followed by a colorimetric reaction, which would produce blue color precipitation on membranes if there is a positive hit. The prototype DNA array contains 29 significant HPV genotypes, including 13 major high-risk (HR)-HPV (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68), and 16 other common HPV genotypes (HPV 6, 11, 26, 40, 42, 43, 44, 53, 54 , 55, 57, 66, 67, 69, 73, and 82). System optimization was carried out using constructed standard HPV plasmids. Clones containing unique L1 fragments of different HPV genotypes were constructed using standard cloning protocol and standard samples were established. PCR was optimized to achieve good amplification for all 29 genotypes. Type-specific probes were designed, tested and selected for specific and sensitive detection of each genotype. Genotype sensitivity of all genotypes was down to 102-103 copy numbers of starting material. The clinical performance of this system was also evaluated using 456 clinical samples from three different sources. Comparison was made between the system developed and commercial tests commonly used, i.e. Digene’s Hybrid Capture 2 (HC2) and Roche Linear Array HPV Genotyping Tests. For comparison to HC2 method, the overall concordances were 87.2%, 93.2% and 94.8%, with kappa value ranged as 0.663, 0.863 and 0.884 respectively, indicating substantial reproducibility and thus, excellent concordant rate with HC2. It also showed good overall compatibility with Roche Linear Array as well, since the strong positive genotypes detected in both methods were almost identical. The system developed is simple and high throughput for rapid in vitro detection and genotyping of HPV infections. The platform is also flexible and could be applied to other infectious pathogens using suitable biomarkers, e.g. for detection of waterborne pathogens, and other common STI pathogens.
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