【摘要】：目的：越來越多的巴爾通體物種感染人類疾病,嚴重的影響人類健康,給患者、家庭和國家造成重大的醫(yī)療衛(wèi)生經(jīng)濟負擔。本課題應用HRM技術建立快速檢測巴爾通體屬通用的方法。在確保檢測出所有巴爾通體物種的情況下,應用雙重HRM方法和雙重實時熒光定量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)測和流行病學調(diào)查等研究提供有效的手段。方法：查閱文獻查找巴爾通體屬的特異引物,應用生物信息學方法查找Bh和Bq特異基因,利用Primer Express 3和Beacon Designer7設計引物和探針。隨后進行常規(guī)PCR擴增,將產(chǎn)物連接到pEASY(?)-T5 Zero克隆載體上制備標準品。優(yōu)化擴增反應的退火溫度、引物探針的濃度和熔解速率；評估各方法的特異性、敏感性及重復性,分析擴增效率和線性關系。結果：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方法檢測巴爾通體屬方法結果顯示特異性很高,只有巴爾通體物種擴增出熒光信號,熔解溫度為81.05℃+0.31,其他陰性對照菌株均未見熒光信號；敏感性實驗結果顯示在20μL的反應體系中,Bh最低檢出限為3.82×101個拷貝,相關系數(shù)R2為0.999,擴增效率E值為98.4%。重復性實驗結果顯示組內(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Bh和Bq結果顯示特異性很高；敏感性實驗結果顯示在20μL的反應體系中,Bh和Bq最低檢出限分別為3.64×101個拷貝和5.62×101個拷貝,此外也顯示出良好的線性關系和擴增效率,其相關系數(shù)R2分別為0.998和0.997,E值分別為102.8%和104.7%。重復性實驗結果顯示組內(nèi)和組間的CV值為0.22%-0.50%和0.50%-1.20%。(3)雙重實時探針法檢測Bh和Bq結果顯示特異性良好；敏感性實驗結果顯示在20μL的反應體系中,Bh和Bq最低檢出限分別為3.64x101個拷貝和3.28×101個拷貝,相關系數(shù)R2分別為0.994和0.998,擴增效率E值分別為100.0%和103.4%；重復性實驗結果顯示組內(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。結論：本研究建立了HRM方法特異性強、靈敏度高、穩(wěn)定性好,可快速地檢測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)測和流行病學調(diào)查等研究提供有效手段。在檢測出所有巴爾通體物種的基礎上,同時也建立了雙重HM技術和雙重實時探針法可快速地檢測區(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：2285518