本文選題：實(shí)時(shí)熒光PCR + armA基因�。� 參考：《中國(guó)疾病預(yù)防控制中心》2015年碩士論文

【摘要】：16S rRNA甲基化酶armA是介導(dǎo)菌株對(duì)氨基糖苷類藥物高水平耐藥的重要機(jī)制,其快速檢測(cè)方法的建立將有助于了解該酶的種屬分布特征和流行傳播規(guī)律。本研究根據(jù)armA基因的特征序列設(shè)計(jì)引物和探針,優(yōu)化反應(yīng)條件,建立armA基因的實(shí)時(shí)熒光PCR檢測(cè)方法,并在多種常見(jiàn)致病菌和糞便模擬標(biāo)本評(píng)價(jià)其靈敏度和特異性。結(jié)果顯示所建立的實(shí)時(shí)熒光PCR檢測(cè)方法對(duì)質(zhì)粒標(biāo)準(zhǔn)品的靈敏度為4.10×101 copy/ml;對(duì)糞便模擬樣品的檢測(cè)下限為1.5×104 cfu/ml。而且該引物探針特異性高,對(duì)其他常見(jiàn)菌種DNA及氨基糖苷類耐藥基因均無(wú)交叉反應(yīng)。應(yīng)用該實(shí)時(shí)熒光PCR檢測(cè)方法,對(duì)收集的846株臨床來(lái)源革蘭氏陰性菌和107株動(dòng)物來(lái)源克雷伯菌菌株中armA基因進(jìn)行檢測(cè)。結(jié)果顯示,臨床來(lái)源菌株armA基因攜帶率為22.0%,其中不動(dòng)菌屬armA基因攜帶率最高,達(dá)到66.5%,其他種屬菌株攜帶率從高到低依次為：沙雷菌屬51.9%,腸桿菌屬27.1%,克雷伯菌屬9.1%,埃希氏菌屬3.3%,假單胞菌屬3.0%。動(dòng)物來(lái)源克雷伯菌屬菌株的armA基因攜帶率為65.4%。應(yīng)用Vitek 2 Compact微生物鑒定藥敏系統(tǒng)對(duì)菌株阿米卡星和慶大霉素兩種抗菌藥物的最小抑菌濃度(MIC)進(jìn)行測(cè)定,結(jié)果表明846株臨床來(lái)源菌株對(duì)阿米卡星和慶大霉素的耐藥率分別為31.3%和60.4%；其中不動(dòng)菌屬對(duì)阿米卡星和慶大霉素的耐藥率最高,分別達(dá)到86.4%和90.9%,其他種屬菌株對(duì)兩種藥物的耐藥率依次為：沙雷菌屬55.6%和55.6%；腸桿菌屬35.4%和62.5%；克雷伯菌屬12.7%和48.1%；埃希氏菌屬7.7%和63.7%；假單胞菌屬10.6%和53.0%。動(dòng)物來(lái)源107株克雷伯菌株對(duì)阿米卡星和慶大霉素的耐藥率分別為74.8%和79.4%。臨床來(lái)源菌株攜帶armA基因與氨基糖苷類耐藥表型的關(guān)系分析顯示：攜帶armA基因的菌株對(duì)阿米卡星耐藥率高達(dá)94.6%,對(duì)慶大霉素耐藥率高達(dá)97.8%,armA基因的攜帶與氨基糖苷類藥物耐藥表型具有很高的一致性。此外,本實(shí)驗(yàn)也對(duì)收集的4株NDM-5陽(yáng)性的大腸埃希菌的特征進(jìn)行了研究。這四株菌來(lái)自國(guó)內(nèi)四個(gè)不同城市的患者,分別導(dǎo)致了患者的尿路感染、傷口感染、細(xì)菌性陰道炎和菌血癥。藥敏檢測(cè)結(jié)果顯示四株菌株對(duì)除氨曲南以外幾乎所有的β-內(nèi)酰胺類藥物均耐藥。多位點(diǎn)序列分析(MLST)結(jié)果顯示：其中兩株屬于ST167型,另外兩株分別是ST2608型和ST2294型。脈沖場(chǎng)凝膠電泳(PFGE)-S1酶切和Southern雜交結(jié)果顯示：盡管4株菌株攜帶的質(zhì)粒數(shù)量和質(zhì)粒大小不盡相同,但攜帶的blaNDM-5基因均位于大小相同的IncX3質(zhì)粒上。上述結(jié)果提示我們,攜帶blaNDM-5基因的ST167型大腸埃希菌和IncX3質(zhì)粒可能介導(dǎo)了blaNDM-5基因在我國(guó)的傳播。
[Abstract]:16s rRNA methylase armA is an important mechanism of high level resistance to aminoglycoside drugs. The rapid detection of 16s rRNA methylase armA will be helpful to understand the characteristics of species distribution and epidemic spread of the enzyme. In this study, primers and probes were designed according to the characteristic sequence of armA gene, and the reaction conditions were optimized. A real-time fluorescence PCR method for detection of armA gene was established, and its sensitivity and specificity were evaluated in a variety of common pathogenic bacteria and stool samples. The results showed that the sensitivity of the established real-time PCR method for plasmid standard was 4.10 脳 101 copy / ml, and the detection limit for fecal simulated samples was 1.5 脳 10 ~ 4 cfur / ml. Moreover, the primer probe was highly specific and had no cross reaction to other common strains of DNA and aminoglycoside resistant genes. The armA gene of 846 strains of Gram-negative bacteria and 107 strains of Klebsiella fauna were detected by real-time fluorescence PCR. The results showed that the carrying rate of armA gene was 22. 0%, among which the armA gene carrying rate of Acinetobacter genus was the highest. The results showed that the carrying rate of other strains was 51.9% of Shareh, 27.1m of Enterobacter, 9.1% of Klebsiella, 3.33% of Ehisiella and 3.0% of Pseudomonas. The armA gene carrying rate of Klebsiella strain was 65.4%. The minimal inhibitory concentration (MIC) of amikacin and gentamicin was determined by Vitek 2 Compact microbiological identification system. The results showed that the resistance rates of 846 clinical strains to amikacin and gentamicin were 31.3% and 60.4%, respectively, and the resistant rates of acinetobacter to amikacin and gentamicin were the highest. The resistance rates of other strains to the two drugs were 55.6% and 55.6% for Shareh, 35.4% and 62.5% for Enterobacter, 12.7% and 48.1% for Klebsiella, 7.7% and 63.7% for Escherichia, 10.6% and 53.0% for Pseudomonas, respectively. The resistance rates of 107 Klebsiella strains to amikacin and gentamicin were 74.8% and 79.4%, respectively. Analysis of the relationship between armA gene and aminoglycoside resistance phenotype of clinical strains showed that the resistance rate of armA gene to amikacin was 94.6, and to gentamicin was 97.8% to aminoglycosides. Drug resistance phenotypes are highly consistent. In addition, the characteristics of four NDM-5 positive Escherichia coli strains were also studied. The four strains were isolated from four different cities in China, leading to urinary tract infection, wound infection, bacterial vaginitis and bacteremia respectively. The results of drug sensitivity test showed that the four strains were resistant to almost all 尾-lactams except aztreonam. The results of multilocus sequence analysis (MLST) showed that two of them belonged to ST167 and the other two were ST2608 and ST2294 respectively. The results of pulsed field gel electrophoresis (PFGE) -S1 digestion and Southern blotting showed that although the number of plasmids and plasmid size of the four strains were different, the blaNDM-5 gene was located on the same size of IncX3 plasmid. These results suggest that ST167 Escherichia coli and IncX3 plasmid carrying blaNDM-5 gene may mediate the transmission of blaNDM-5 gene in China.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R440

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