【摘要】：目的：越來越多的巴爾通體物種感染人類疾病,嚴(yán)重的影響人類健康,給患者、家庭和國(guó)家造成重大的醫(yī)療衛(wèi)生經(jīng)濟(jì)負(fù)擔(dān)。本課題應(yīng)用HRM技術(shù)建立快速檢測(cè)巴爾通體屬通用的方法。在確保檢測(cè)出所有巴爾通體物種的情況下,應(yīng)用雙重HRM方法和雙重實(shí)時(shí)熒光定量PCR探針法建立快速鑒定區(qū)分引起心內(nèi)膜炎常見的兩種病原體(Bq和Bh),為臨床疾病的早期診斷、監(jiān)測(cè)和流行病學(xué)調(diào)查等研究提供有效的手段。方法：查閱文獻(xiàn)查找巴爾通體屬的特異引物,應(yīng)用生物信息學(xué)方法查找Bh和Bq特異基因,利用Primer Express 3和Beacon Designer7設(shè)計(jì)引物和探針。隨后進(jìn)行常規(guī)PCR擴(kuò)增,將產(chǎn)物連接到pEASY(?)-T5 Zero克隆載體上制備標(biāo)準(zhǔn)品。優(yōu)化擴(kuò)增反應(yīng)的退火溫度、引物探針的濃度和熔解速率；評(píng)估各方法的特異性、敏感性及重復(fù)性,分析擴(kuò)增效率和線性關(guān)系。結(jié)果：優(yōu)化的退火溫度均為60℃,引物濃度均為300 nmoL/L,探針濃度均為200nmoL/L和熔解速率為0.2℃/s。(1)HRM方法檢測(cè)巴爾通體屬方法結(jié)果顯示特異性很高,只有巴爾通體物種擴(kuò)增出熒光信號(hào),熔解溫度為81.05℃+0.31,其他陰性對(duì)照菌株均未見熒光信號(hào)；敏感性實(shí)驗(yàn)結(jié)果顯示在20μL的反應(yīng)體系中,Bh最低檢出限為3.82×101個(gè)拷貝,相關(guān)系數(shù)R2為0.999,擴(kuò)增效率E值為98.4%。重復(fù)性實(shí)驗(yàn)結(jié)果顯示組內(nèi)和組間的CV值分別為0.30%-0.62%和0.29%-0.36%。(2)雙重HRM方法檢測(cè)Bh和Bq結(jié)果顯示特異性很高；敏感性實(shí)驗(yàn)結(jié)果顯示在20μL的反應(yīng)體系中,Bh和Bq最低檢出限分別為3.64×101個(gè)拷貝和5.62×101個(gè)拷貝,此外也顯示出良好的線性關(guān)系和擴(kuò)增效率,其相關(guān)系數(shù)R2分別為0.998和0.997,E值分別為102.8%和104.7%。重復(fù)性實(shí)驗(yàn)結(jié)果顯示組內(nèi)和組間的CV值為0.22%-0.50%和0.50%-1.20%。(3)雙重實(shí)時(shí)探針法檢測(cè)Bh和Bq結(jié)果顯示特異性良好；敏感性實(shí)驗(yàn)結(jié)果顯示在20μL的反應(yīng)體系中,Bh和Bq最低檢出限分別為3.64x101個(gè)拷貝和3.28×101個(gè)拷貝,相關(guān)系數(shù)R2分別為0.994和0.998,擴(kuò)增效率E值分別為100.0%和103.4%；重復(fù)性實(shí)驗(yàn)結(jié)果顯示組內(nèi)和組間的CV值分別為0.19%-0.53%和0.49%-1.66%。建立的方法敏感性均比常規(guī)PCR提高了100倍。結(jié)論：本研究建立了HRM方法特異性強(qiáng)、靈敏度高、穩(wěn)定性好,可快速地檢測(cè)出所有的巴爾通體物種,為巴爾通體所引起的CSD、戰(zhàn)壕熱、心內(nèi)膜炎、BA和卡瑞恩病等一系列疾病的早期快速診斷、監(jiān)測(cè)和流行病學(xué)調(diào)查等研究提供有效手段。在檢測(cè)出所有巴爾通體物種的基礎(chǔ)上,同時(shí)也建立了雙重HM技術(shù)和雙重實(shí)時(shí)探針法可快速地檢測(cè)區(qū)分引起心內(nèi)膜炎常見的病原體Bq和Bh。
[Abstract]:Objective: more and more Bartonella species are infected with human diseases, which seriously affect human health. In this paper, HRM technique is used to establish a universal method for rapid detection of Bartonella. To ensure the detection of all Bartonella species, double HRM and double real-time fluorescence quantitative PCR probes were used to establish a rapid identification of two common pathogens causing endocarditis (Bq and Bh), as early diagnosis of clinical diseases). Studies such as surveillance and epidemiological investigation provide an effective means. Methods: the specific primers of Bartonella were searched and the specific genes of Bh and Bq were searched by bioinformatics. Primers and probes were designed by Primer Express 3 and Beacon Designer7. The product was then amplified by conventional PCR and ligated to pEASY (?)-T _ 5 Zero clone vector to prepare the standard product. The annealing temperature of amplification reaction, the concentration of primer probe and melting rate were optimized, the specificity, sensitivity and repeatability of each method were evaluated, and the relationship between amplification efficiency and linearity was analyzed. Results: the optimized annealing temperature was 60 鈩，

本文編號(hào)：2285519