耐萬古霉素腸球菌檢測基因芯片方法的建立和驗(yàn)證
發(fā)布時(shí)間：2018-10-26 06:43
【摘要】：腸球菌屬于革蘭氏陽性球菌,它廣泛分布于自然環(huán)境及人和動(dòng)物的消化道內(nèi),其致病力較弱,只有在宿主組織寄殖,耐機(jī)體非特異及免疫防御機(jī)制,并引起病理改變才能導(dǎo)致感染。但是由于近年來在臨床中廣譜抗生素尤其是萬古霉素的大量使用以及在使用中的不合理現(xiàn)象,導(dǎo)致了耐萬古霉素腸球菌(VRE)的出現(xiàn),從而引起了全世界范圍內(nèi)的廣泛關(guān)注。近年來大量研究顯示,由于VRE更容易引起院內(nèi)感染及播散,其檢測、鑒別與診斷也成為了醫(yī)務(wù)工作者及研究者們所面臨的重要問題。目前常用VRE的檢測方法為細(xì)菌學(xué)方法,其使用耗時(shí)且無法準(zhǔn)確辨別腸球菌的菌種類型;并且腸球菌屬中不同種細(xì)菌對(duì)抗菌藥物的耐藥性存在差異,其也無法區(qū)分VRE的耐藥基因。相對(duì)于藥敏實(shí)驗(yàn)的不足,本研究選取了2種常見的腸球菌,旨在應(yīng)用基因芯片檢測技術(shù)研制可以區(qū)分VRE種類及耐藥基因型的基因芯片,為深入開展基因芯片檢測VRE更廣泛應(yīng)用于臨床奠定基礎(chǔ)。我們以16S rDNA基因作為檢測靶片段,耐萬古霉素特異性基因及腸球菌的特異性基因作為靶序列,根據(jù)GeneBank中查找到的2種常見腸球菌特異性基因序列(ddl)及萬古霉素耐藥基因(VanA,VanB),利用BLAST等比對(duì)軟件,設(shè)計(jì)與合成用于檢測腸球菌的特異性基因及耐藥基因的引物和探針,制備耐萬古霉素腸球菌檢測基因芯片。利用多重不對(duì)稱PCR法擴(kuò)增樣品中的特異性基因與耐藥基因片段,標(biāo)記產(chǎn)物與基因芯片上的探針雜交,經(jīng)清洗、化學(xué)發(fā)光法顯色后進(jìn)行結(jié)果分析。在優(yōu)化的多重PCR體系、雜交反應(yīng)和化學(xué)發(fā)光法檢測條件下,評(píng)價(jià)芯片的特異性、靈敏性和重復(fù)性等性能指標(biāo),并應(yīng)用于臨床樣本的檢測。最后本研究所選取的靶耐藥基因和腸球菌特異性基因?yàn)槟腿f古霉素A基因(VanA)、耐萬古霉素B基因(VanB)、腸球菌特異性基因(ddl),16S rDNA基因作為內(nèi)對(duì)照,共篩選出1對(duì)通用引物、4對(duì)特異性引物和1條細(xì)菌通用探針、4條特異性檢測探針。確定了五重PCR體系。確定了每種探針的cutoff值。檢測耐萬古霉素腸球菌的靈敏度為103cfu/mL,批間和批內(nèi)變異系數(shù)CV值均小于15%。對(duì)40例臨床樣本進(jìn)行檢測,結(jié)果與常規(guī)培養(yǎng)法培養(yǎng)結(jié)果基本一致。因此綜上所述本研究建立的耐萬古霉素腸球菌基因芯片檢測方法具有高靈敏性、高特異性且高通量的優(yōu)點(diǎn),基本能夠區(qū)分腸球菌類型及相關(guān)耐藥的基因型。該方法可為臨床中耐萬古霉素腸球菌的檢測、個(gè)體化選擇用藥、監(jiān)測預(yù)后及院內(nèi)感染提供了更新的實(shí)驗(yàn)方法,其結(jié)果可靠,并降低了操作難度及成本,對(duì)于減少資源浪費(fèi)和避免抗生素帶來更多不良反應(yīng)的風(fēng)險(xiǎn)有重要意義。
[Abstract]:Enterococcus belongs to Gram-positive cocci. It is widely distributed in the natural environment and the digestive tract of human and animal. Its pathogenicity is weak. And cause pathological changes to cause infection. However, in recent years, the widespread use and unreasonable use of broad spectrum antibiotics, especially vancomycin, has led to the emergence of vancomycin resistant Enterococcus (VRE), which has attracted worldwide attention. In recent years, a large number of studies have shown that because VRE is more likely to cause nosocomial infection and spread, its detection, identification and diagnosis have become an important problem for medical workers and researchers. At present, the commonly used VRE detection method is bacteriological method, which is time-consuming and can not accurately identify the type of Enterococcus, and the resistance of different strains of Enterococcus to antimicrobial agents is different, and it can not distinguish the drug-resistant genes of VRE. Compared with the deficiency of drug sensitivity test, two common enterococci were selected in this study. The purpose of this study was to develop a gene chip that could distinguish VRE species from drug resistant genotypes by using gene chip detection technology. It lays a foundation for further application of gene chip detection of VRE in clinical practice. We used 16s rDNA gene as target fragment, vancomycin-resistant specific gene and Enterococcus specific gene as target sequence. According to (ddl) and vancomycin resistant gene (VanA,VanB), two common enterococci specific gene sequences were found in GeneBank. BLAST et al. Primers and probes were designed and synthesized to detect the specific genes and drug resistance genes of Enterococcus enterococcus, and the vancomycin resistant Enterococcus microarray was prepared. The specific gene and drug resistance gene fragments were amplified by multiple asymmetric PCR method. The labeled products were hybridized with probes on the gene chip. The results were analyzed by cleaning and chemiluminescence method. In the optimized multiplex PCR system, hybridization reaction and chemiluminescence assay, the specificity, sensitivity and repeatability of the chip were evaluated and applied to the detection of clinical samples. Finally, the target resistance gene and Enterococcus specific gene were selected as internal control because vancomycin A gene, (VanA), gene, vancomycin B gene, (VanB), gene, Enterococcus specific gene (ddl), 16s rDNA gene, were selected as internal control. A pair of universal primers, four pairs of specific primers, one universal bacterial probe and four specific detection probes were screened. The five-fold PCR system is determined. The cutoff value of each probe was determined. The sensitivity of detection of vancomycin resistant Enterococcus was 103 cfur / mL, and the coefficient of variation (CV) was less than 15%. The clinical samples of 40 cases were detected and the results were consistent with the results of conventional culture method. In conclusion, the vancomycin resistant Enterococcus gene chip assay has the advantages of high sensitivity, high specificity and high throughput, and can basically distinguish Enterococcus and related drug resistant genotypes. This method can provide a new experimental method for the detection of vancomycin resistant Enterococcus, individualized drug selection, prognosis monitoring and nosocomial infection. The results are reliable, and the operation difficulty and cost are reduced. It is important to reduce the waste of resources and avoid the risk of more adverse reactions caused by antibiotics.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
本文編號(hào)：2294887
[Abstract]:Enterococcus belongs to Gram-positive cocci. It is widely distributed in the natural environment and the digestive tract of human and animal. Its pathogenicity is weak. And cause pathological changes to cause infection. However, in recent years, the widespread use and unreasonable use of broad spectrum antibiotics, especially vancomycin, has led to the emergence of vancomycin resistant Enterococcus (VRE), which has attracted worldwide attention. In recent years, a large number of studies have shown that because VRE is more likely to cause nosocomial infection and spread, its detection, identification and diagnosis have become an important problem for medical workers and researchers. At present, the commonly used VRE detection method is bacteriological method, which is time-consuming and can not accurately identify the type of Enterococcus, and the resistance of different strains of Enterococcus to antimicrobial agents is different, and it can not distinguish the drug-resistant genes of VRE. Compared with the deficiency of drug sensitivity test, two common enterococci were selected in this study. The purpose of this study was to develop a gene chip that could distinguish VRE species from drug resistant genotypes by using gene chip detection technology. It lays a foundation for further application of gene chip detection of VRE in clinical practice. We used 16s rDNA gene as target fragment, vancomycin-resistant specific gene and Enterococcus specific gene as target sequence. According to (ddl) and vancomycin resistant gene (VanA,VanB), two common enterococci specific gene sequences were found in GeneBank. BLAST et al. Primers and probes were designed and synthesized to detect the specific genes and drug resistance genes of Enterococcus enterococcus, and the vancomycin resistant Enterococcus microarray was prepared. The specific gene and drug resistance gene fragments were amplified by multiple asymmetric PCR method. The labeled products were hybridized with probes on the gene chip. The results were analyzed by cleaning and chemiluminescence method. In the optimized multiplex PCR system, hybridization reaction and chemiluminescence assay, the specificity, sensitivity and repeatability of the chip were evaluated and applied to the detection of clinical samples. Finally, the target resistance gene and Enterococcus specific gene were selected as internal control because vancomycin A gene, (VanA), gene, vancomycin B gene, (VanB), gene, Enterococcus specific gene (ddl), 16s rDNA gene, were selected as internal control. A pair of universal primers, four pairs of specific primers, one universal bacterial probe and four specific detection probes were screened. The five-fold PCR system is determined. The cutoff value of each probe was determined. The sensitivity of detection of vancomycin resistant Enterococcus was 103 cfur / mL, and the coefficient of variation (CV) was less than 15%. The clinical samples of 40 cases were detected and the results were consistent with the results of conventional culture method. In conclusion, the vancomycin resistant Enterococcus gene chip assay has the advantages of high sensitivity, high specificity and high throughput, and can basically distinguish Enterococcus and related drug resistant genotypes. This method can provide a new experimental method for the detection of vancomycin resistant Enterococcus, individualized drug selection, prognosis monitoring and nosocomial infection. The results are reliable, and the operation difficulty and cost are reduced. It is important to reduce the waste of resources and avoid the risk of more adverse reactions caused by antibiotics.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
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相關(guān)期刊論文 前2條
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