發(fā)布時(shí)間：2018-03-03 16:07

  本文選題：人乳頭瘤病毒　切入點(diǎn)：膠體金-探針　出處：《西北大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 研究背景：目前已知宮頸癌的發(fā)病主要與人乳頭瘤病毒(HPV)的高癌型感染有關(guān)。據(jù)統(tǒng)計(jì)全球每年有25萬以上的婦女死于宮頸癌,宮頸癌是女性的第二位高發(fā)癌。隨著每年宮頸癌和生殖器疣的患病率增加,對HPV感染的篩查有重要的意義。為了滿足我國現(xiàn)階段宮頸癌的防治需要,對婦女高危人群中宮頸糜爛患者和外生殖器部位尖銳濕疣的HPV感染分型和定量的流行病學(xué)調(diào)查與研究,對HPV的傳播、宮頸癌的癌前診斷及預(yù)防提供重要依據(jù),本項(xiàng)目的目的在于構(gòu)建高通量、快速、靈敏且特異的HPV基因分型檢測芯片系統(tǒng)。 方法：本研究以目視化基因芯片為平臺(tái),引入多重不對稱PCR及膠體金銀染著色技術(shù),以13種高、低危人乳頭瘤病毒為模板,建立了可目視化基因分型的新方法。我們將樣本病毒基因提取出來之后,利用通用引物GP5/GP6對基因組進(jìn)行不對稱擴(kuò)增；設(shè)計(jì)兩種探針,一種稱之為捕獲探針,在探針的5’末端進(jìn)行氨基修飾,使其能和玻片連接,捕獲探針用于和目的片段結(jié)合；另外一種探針稱之為檢測探針,在探針的3’端進(jìn)行巰基修飾,使其能和膠體金形成共價(jià)連接,檢測探針為非限制性引物的反義鏈可以和目的片段互補(bǔ)結(jié)合；在雜交過程中捕獲探針和檢測探針同時(shí)與目的片段相結(jié)合,膠體金就間接的標(biāo)記到目的片段上,通過顯色液銀染顯色,就會(huì)在芯片上出現(xiàn)一個(gè)可以肉眼觀測的黑色或灰色斑點(diǎn),通過平板掃描儀就可以對結(jié)果進(jìn)行記錄和分析。 結(jié)果： 1.在檢測中我們引入了β球蛋白基因作為系統(tǒng)的陽性指控。在對100例臨床樣本的檢測種目視化芯片檢測法與細(xì)胞學(xué)檢測法的陽性率間無顯著性差異(P0.05)。 2.檢測探針濃度優(yōu)化為1μM-5μM之間做為后期實(shí)驗(yàn)的探針最佳點(diǎn)樣濃度。 3.在不對稱引物比例上經(jīng)過試驗(yàn)驗(yàn)證限制性引物和非限制性引物比例為1：20時(shí)效果最好,單鏈產(chǎn)物最多。 4.通過軟件計(jì)算的數(shù)據(jù)可以得出在病毒模板稀釋10-9時(shí)還有信號產(chǎn)生,銀染時(shí)間為10min 5.穩(wěn)定1 mL lOnm納米金(A520=0.842)的巰基修飾探針的用量為0.4μg。
[Abstract]:Background: it is known that the incidence of cervical cancer is mainly related to the human papillomavirus (HPV) high carcinomatous infection. According to statistics, more than 250,000 women die of cervical cancer every year in the world. Cervical cancer is the second most common cancer in women. With the increasing incidence of cervical cancer and genital warts every year, screening for HPV infection is of great significance. In order to provide important basis for the transmission of HPV and the precancerous diagnosis and prevention of cervical cancer, the types and quantitative epidemiological investigation of HPV infection in cervical erosion patients and condyloma acuminatum of external genitalia in high-risk women were investigated. The aim of this project is to construct a high throughput, fast, sensitive and specific HPV genotyping detection chip system. Methods: using visual microarray as a platform, multiple asymmetric PCR and colloidal gold and silver staining techniques were introduced. Thirteen high and low risk human papillomavirus (HPVs) were used as templates. A new method of visual genotyping was established. After we extracted the virus gene from the sample, we used universal primer GP5/GP6 to amplify the genome asymmetrically, and designed two kinds of probes, one of which is called capture probe. The probe is modified with amino groups at the 5'end of the probe to enable it to connect to the glass slide, and the probe is captured for binding to the target fragment. The other probe is called the detection probe, which is modified with sulfhydryl groups at the 3 'end of the probe. It can be covalently connected with colloidal gold, and the antisense strand of the detection probe, which is a non-restrictive primer, can be complementary to the target fragment, and the capture probe and the detection probe can be combined with the target fragment simultaneously during hybridization. Colloidal gold is indirectly labeled to the target fragment, and a black or gray spot can be observed on the chip by silver staining, and the results can be recorded and analyzed by a flat plate scanner. Results:. 1. We introduced the 尾 globulin gene as a systematic positive charge. There was no significant difference in the positive rate between visual microarray and cytology in 100 clinical samples. 2. The detection probe concentration was optimized between 1 渭 M-5 渭 M as the best sample concentration in the later experiment. 3. When the proportion of restriction primer and unrestricted primer was 1:20, the best effect was obtained and the single strand product was the most. 4. The data calculated by the software can be obtained when the virus template is diluted 10-9, and the silver staining time is 10min. 5. The amount of thiol modified probe was 0.4 渭 g.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R373

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本文編號：1561729