幽门螺杆菌的培养鉴定及其基因分型方法学的建立

发布时间：2018-07-10 05:06

本文选题：幽门螺杆菌 + 基因分型　；参考：《浙江大学》2007年硕士论文

【摘要】： 背景和目的幽门螺杆菌(Helicobacter pylori，Hp)是目前世界上最常见的人类感染细菌之一，全球约50％左右的人群均感染过Hp，仅次于龋齿，中国的感染率约为60％。大量的研究已经表明，Hp是慢性胃炎、消化性溃疡的主要致病因素，并与胃癌、胃MALT淋巴瘤的发生也密切相关。1994年世界卫生组织将其列为第一类生物致癌因子。但是在研究中人们发现，并非所有Hp感染者都会患胃肠疾病，这就说明只有部分Hp与疾病相关，且有可能不同类型的Hp与不同的疾病相关，于是有学者就开始进行Hp型别与疾病关系的研究。由于Hp是一种表型特征一致、但基因变异很高的细菌，故表型分型方法的分型效率比较低，许多分子分型方法应运而生。在这些分子分型方法中，PCR产物分型因其操作简单、方便、所需模板量少等优点而在实际中得到广泛使用。故本研究建立一种直接PCR方法针对Hp三类主要毒力因子ureA、cagA和vacA基因及vacA基因亚型进行综合分析分型，希望从Hp的基因水平上来探讨其与不同上消化道疾病之间的关系。另外，由于Hp生长条件要求苛刻，培养起来即复杂又困难，因此本研究还对Hp的培养及鉴定方法进行了一定的摸索。材料与方法标本来源于在宁波镇海龙赛医院因上消化道症状进行胃镜检查，采取胃窦黏膜，通过对患者胃窦粘膜组织进行增菌、分离培养、挑取可疑菌落涂片镜检，并对其进行有关Hp生化反应鉴定；对分离鉴定的Hp菌株进行DNA提取；采用PCR扩增特异性ureA基因鉴定菌株。采用特定引物对各菌株基因型进行PCR扩增，扩增产物在2％琼脂糖凝胶中进行电泳；在凝胶分析系统中观察电泳并照相。扩增产物通过克隆测序，BLAST同源分析验证。结果 1．本研究146例胃窦粘膜活检标本中，培养鉴定84例Hp阳性菌株，培养Hp阳性率为57.53％。 2．培养鉴定的84例Hp菌株中，ureA基因的阳性率100％(84／84)，cagA基因的阳性率为90.48％(76／84)，vacA基因的阳性率95.24％(80／84)，cagA、vacA在胃癌中的阳性率均较低，与胃炎、消化性溃疡比较有显著性差异(p＜0.05)。 3．PCR产物经过BLAST同源分析，PCR鉴定基因型结果与测序结果一致。 4．VacAm2基因的阳性率为79.76％(67／84)，vacAmla基因的阳性率20.24％(17／84)，，vacAmlb基因8.33％(7／84)，vacAmlbm24.76％(4／84)，比较分析各基因组Hp在各疾病组间阳性率，vacAm2在胃癌中的阳性率最低，与胃炎、消化性溃疡比较有显著性差异(p＜0.05)，vacAm2在消化性溃疡中的阳性率与胃炎比较无显著性差异(p＞0.05)，其余3个vacA基因亚型在胃炎、消化性溃疡、胃癌阳性率比较均无显著性差异(p均＞0.05)。 5．VacAm2亚型在胃炎、消化性溃疡的阳性率较高(77.78％，86.44％)，与cagA(83.33％，96.78％)比较差异无显著性(p＞0.05)，VacA其他三个亚型在胃炎、消化性溃疡的阳性率均较低，与cagA比较差异有显著性(p均＜0.05)。结论 1．本试验Hp培养鉴定改良了Hp培养的配方，使得培养更简单易行，阳性率较目前常规应用更高，为药敏试验创造了良好的基础； 2．本实验组Hp相关性上消化道疾病患者感染的Hp以cagA阳性、VacAm2亚型为优势； 3．本研究运用PCR技术对幽门螺杆菌ureA、cagA、vacA三个基因及vacA 基因亚型分型方法的建立是快速而准确的，并用分子流行病方法表明了三种胃部疾病与基因型别的关系，是一套行之有效的分型系统。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

【参考文献】