本文選題：幽門螺桿菌 + 基因分型��； 參考：《浙江大學》2007年碩士論文

【摘要】： 背景和目的 幽門螺桿菌(Helicobacter pylori，Hp)是目前世界上最常見的人類感染細菌之一，全球約50％左右的人群均感染過Hp，僅次于齲齒，中國的感染率約為60％。大量的研究已經表明，Hp是慢性胃炎、消化性潰瘍的主要致病因素，并與胃癌、胃MALT淋巴瘤的發(fā)生也密切相關。1994年世界衛(wèi)生組織將其列為第一類生物致癌因子。但是在研究中人們發(fā)現，并非所有Hp感染者都會患胃腸疾病，這就說明只有部分Hp與疾病相關，且有可能不同類型的Hp與不同的疾病相關，于是有學者就開始進行Hp型別與疾病關系的研究。由于Hp是一種表型特征一致、但基因變異很高的細菌，故表型分型方法的分型效率比較低，許多分子分型方法應運而生。在這些分子分型方法中，PCR產物分型因其操作簡單、方便、所需模板量少等優(yōu)點而在實際中得到廣泛使用。故本研究建立一種直接PCR方法針對Hp三類主要毒力因子ureA、cagA和vacA基因及vacA基因亞型進行綜合分析分型，希望從Hp的基因水平上來探討其與不同上消化道疾病之間的關系。另外，由于Hp生長條件要求苛刻，培養(yǎng)起來即復雜又困難，因此本研究還對Hp的培養(yǎng)及鑒定方法進行了一定的摸索。 材料與方法 標本來源于在寧波鎮(zhèn)海龍賽醫(yī)院因上消化道癥狀進行胃鏡檢查，采取胃竇黏膜，通過對患者胃竇粘膜組織進行增菌、分離培養(yǎng)、挑取可疑菌落涂片鏡檢，并對其進行有關Hp生化反應鑒定；對分離鑒定的Hp菌株進行DNA提取；采用PCR擴增特異性ureA基因鑒定菌株。采用特定引物對各菌株基因型進行PCR擴增，擴增產物在2％瓊脂糖凝膠中進行電泳；在凝膠分析系統(tǒng)中觀察電泳并照相。擴增產物通過克隆測序，BLAST同源分析驗證。 結果 1．本研究146例胃竇粘膜活檢標本中，培養(yǎng)鑒定84例Hp陽性菌株，培養(yǎng)Hp陽性率為57.53％。 2．培養(yǎng)鑒定的84例Hp菌株中，ureA基因的陽性率100％(84／84)，cagA基因的陽性率為90.48％(76／84)，vacA基因的陽性率95.24％(80／84)，cagA、vacA在胃癌中的陽性率均較低，與胃炎、消化性潰瘍比較有顯著性差異(p＜0.05)。 3．PCR產物經過BLAST同源分析，PCR鑒定基因型結果與測序結果一致。 4．VacAm2基因的陽性率為79.76％(67／84)，vacAmla基因的陽性率20.24％(17／84)，，vacAmlb基因8.33％(7／84)，vacAmlbm24.76％(4／84)，比較分析各基因組Hp在各疾病組間陽性率，vacAm2在胃癌中的陽性率最低，與胃炎、消化性潰瘍比較有顯著性差異(p＜0.05)，vacAm2在消化性潰瘍中的陽性率與胃炎比較無顯著性差異(p＞0.05)，其余3個vacA基因亞型在胃炎、消化性潰瘍、胃癌陽性率比較均無顯著性差異(p均＞0.05)。 5．VacAm2亞型在胃炎、消化性潰瘍的陽性率較高(77.78％，86.44％)，與cagA(83.33％，96.78％)比較差異無顯著性(p＞0.05)，VacA其他三個亞型在胃炎、消化性潰瘍的陽性率均較低，與cagA比較差異有顯著性(p均＜0.05)。 結論 1．本試驗Hp培養(yǎng)鑒定改良了Hp培養(yǎng)的配方，使得培養(yǎng)更簡單易行，陽性率較目前常規(guī)應用更高，為藥敏試驗創(chuàng)造了良好的基礎； 2．本實驗組Hp相關性上消化道疾病患者感染的Hp以cagA陽性、VacAm2亞型為優(yōu)勢； 3．本研究運用PCR技術對幽門螺桿菌ureA、cagA、vacA三個基因及vacA 基因亞型分型方法的建立是快速而準確的，并用分子流行病方法表明了三種胃部疾病與基因型別的關系，是一套行之有效的分型系統(tǒng)。
[Abstract]:Background and purpose
Helicobacter pylori (Hp) is one of the most common human infected bacteria in the world. Around 50% of the world is infected with Hp, second only to dental caries. The infection rate in China is about 60%., which has shown that Hp is the main pathogenic factor of chronic gastritis, digestive ulceration, and gastric cancer and gastric MALT lymphoma. It is also closely related to.1994 as the first class of biological carcinogens in.1994. But in the study, it is found that not all Hp infected people suffer from gastrointestinal diseases, which means that only part of the Hp is associated with the disease and that different types of Hp may be associated with different diseases, and some scholars begin to carry out Hp types. The study of the relationship with disease. Because Hp is a kind of bacteria with high phenotypic characteristics but very high genetic variation, the typing efficiency of phenotypic typing is low and many molecular typing methods emerge. In these molecular typing methods, the PCR product typing is widely used in practice because of its simple operation, convenience and less template. In this study, a direct PCR method was established for the comprehensive analysis of Hp three major virulence factors ureA, cagA and vacA genes and vacA gene subtypes. The relationship between them and different upper digestive tract diseases was discussed from the gene level of Hp. In addition, due to the harsh conditions of Hp growth conditions, the culture was complicated and sleepy. Therefore, this study also explored the cultivation and identification methods of Hp.
Materials and methods
The specimens were derived from gastroscopy in the upper gastrointestinal tract in Hailong hospital, Ningbo, Ningbo. The gastric antrum mucosa was used to isolate and culture the mucosa of the gastric antrum, and to identify the biochemical reaction of the suspected bacterial colonies. The Hp strains isolated and identified by DNA were extracted and PCR amplification was used. Heterosexual ureA gene identification strains were amplified by specific primers, and the amplified products were amplified in 2% agarose gel. The electrophoresis was observed and photographed in the gel analysis system. The amplified products were cloned and sequenced and BLAST homologous analysis was proved.
Result
1. in this study, 146 cases of gastric antral mucosa biopsy specimens were cultured and identified 84 Hp positive strains. The positive rate of culture Hp was 57.53%.
2. of the 84 Hp strains identified by culture, the positive rate of ureA gene was 100% (84 / 84), the positive rate of cagA gene was 90.48% (76 / 84), the positive rate of vacA gene was 95.24% (80 / 84), the positive rate of cagA and vacA in gastric cancer was lower, compared with gastritis and peptic ulcer (P < 0.05).
3.PCR products were identified by BLAST homology analysis, and PCR genotypes were identical with the sequencing results.
The positive rate of 4.VacAm2 gene was 79.76% (67 / 84), the positive rate of vacAmla gene was 20.24% (17 / 84), the vacAmlb gene was 8.33% (7 / 84), and vacAmlbm24.76% (4 / 84). The positive rate of Hp in each disease group was compared. The positive rate of vacAm2 in gastric cancer was the lowest, and there was a significant difference from gastritis and peptic ulcer (P < 0.05), VA. The positive rate of cAm2 in peptic ulcer was not significantly different from that of gastritis (P > 0.05). The other 3 vacA subtypes were not significantly different in gastritis, peptic ulcers and gastric cancer (P > 0.05).
The positive rate of 5.VacAm2 subtype in gastritis and peptic ulcer was higher (77.78%, 86.44%), compared with cagA (83.33%, 96.78%), there was no significant difference (P > 0.05). The other three subtypes of VacA were lower in gastritis and peptic ulcers, compared with cagA (P < 0.05).
conclusion
1. the test of Hp culture has improved the formula of Hp culture, which makes the culture more simple and easier, the positive rate is higher than the current routine application, which creates a good foundation for the drug sensitivity test.
2. in the experimental group, the Hp of Hp related upper gastrointestinal disease patients was cagA positive, and VacAm2 subtype was the advantage.
3. in this study, three genes and vacA of Helicobacter pylori ureA, cagA, vacA were detected by PCR.
The establishment of the genotyping method is rapid and accurate, and the relationship between three kinds of stomach diseases and genotypes is demonstrated by the molecular epidemiological method. It is an effective system of typing.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R378

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