【摘要】：【目的】以煙曲霉內(nèi)轉(zhuǎn)錄間隔區(qū)(ITSD)為靶序列,篩選煙曲霉菌特異性的引物對,應(yīng)用重組酶介導(dǎo)的等溫核酸擴(kuò)增(RPA)技術(shù)建立快速檢測煙曲霉的新方法并對該檢測方法進(jìn)行方法學(xué)優(yōu)化�！痉椒ā�(1)以煙曲霉菌的易變區(qū)內(nèi)轉(zhuǎn)錄間隔區(qū)為靶序列,先用Primer-5軟件在不同區(qū)間設(shè)計三條上游引物和三條下游引物,交叉配對組成不同引物對,然后將引物逐一進(jìn)行Primer-blast,篩選煙曲霉的特異性引物。(2)應(yīng)用機(jī)械性玻璃珠打擊裂解法抽提獲得煙曲霉標(biāo)準(zhǔn)菌株的DNA。(3)通過優(yōu)化RPA檢測煙曲霉的引物長度、產(chǎn)物長度、擴(kuò)增時間及擴(kuò)增溫度等檢測條件,建立檢測煙曲霉的新方法。(4)通過交叉擴(kuò)增實(shí)驗(yàn)及檢測不同濃度煙曲霉DNA樣品來評估RPA檢測煙曲霉菌的敏感性和特異性。(5)通過對21株臨床分離的人致病性曲霉菌菌株的檢測來評估RPA技術(shù)的臨床檢測性能。(6)通過RPA與PCR同時進(jìn)行敏感性、特異性、臨床分離曲霉菌檢測試驗(yàn),對RPA與PCR技術(shù)進(jìn)行方法學(xué)比較�！窘Y(jié)果】(1)篩選得到煙曲霉特異性的引物為上游位于ITSD1、下游位于ITSD2、擴(kuò)增產(chǎn)物為489bp的引物。引物序列為:上游引物:5'-GGTCCAACCTCCCACCCGTGTCTATC-3',下游引物:5'-TTAGAAAAATAAAGTTGGGTGTCGGC-3'(2)通過機(jī)械性玻璃珠打擊裂解法抽提獲得的煙曲霉基因組DNA在純度和的量上能夠滿足核酸擴(kuò)增需要,且簡便、快速。(3)RPA技術(shù)檢測煙曲霉菌的優(yōu)化反應(yīng)條件為:引物長度范圍為18-32bp;擴(kuò)增產(chǎn)物長度范圍為200-700bp;擴(kuò)增溫度為37-42℃;擴(kuò)增時間為15-40min。(4)RPA特異性和敏感性:不同曲霉菌種交叉擴(kuò)增實(shí)驗(yàn)顯示,RPA只在煙曲霉菌擴(kuò)增中有明顯的特異性條帶,而對黃曲霉、土曲霉、黑曲霉、構(gòu)巢曲霉、中國紅酵母均無擴(kuò)增。在以瓊脂糖凝膠電泳為檢測平臺時,RPA可以檢測到最低濃度為100pg/μl的煙曲霉DNA樣品。(5)21株臨床分離的人致病性曲霉菌檢測實(shí)驗(yàn)中,RPA對18株煙曲霉分離菌株的擴(kuò)增均為陽性,而3株黃曲霉擴(kuò)增均為陰性。(6)通過對RPA和PCR檢測煙曲霉方法學(xué)的比較,RPA和PCR一樣具有很高的敏感性和很強(qiáng)的特異性�！窘Y(jié)論】本研究通過Primer-Blast篩選針對煙曲霉ITS1-ITS2靶DNA序列的特異性引物。在優(yōu)化反應(yīng)條件下,應(yīng)用此特異性引物建立的RPA檢測煙曲霉方法可以在較低的恒溫條件下對煙曲霉菌DNA進(jìn)行有效擴(kuò)增,具有簡便、快速、成本低的特點(diǎn),而且與PCR技術(shù)具有相同的敏感性和和特異性,適合用于煙曲霉菌的快速檢測和鑒定。
[Abstract]:[objective] to screen the specific primer pairs of Aspergillus fumigatus using (ITSD) as the target sequence. A new method for rapid detection of Aspergillus fumigatus was established by isothermal amplification of nucleic acid (RPA) mediated by recombinant enzyme. [methods] (1) the transcriptional spacer region of variable region of Aspergillus fumigatus was used as the target sequence. First, three upstream primers and three downstream primers were designed with Primer-5 software in different regions. Then Primer-blast, was used to screen the specific primers of Aspergillus fumigatus one by one. (2) the DNA. of Aspergillus fumigatus standard strain was extracted by mechanical glass bead cracking method. (3) the length of primer and product of Aspergillus fumigatus were optimized by RPA. Amplification time and temperature, etc., A new method for the detection of Aspergillus fumigatus was established. (4) the sensitivity and specificity of RPA for detection of Aspergillus fumigatus were evaluated by cross-amplification test and detection of different concentrations of Aspergillus fumigatus DNA. (5) the sensitivity and specificity of RPA for detection of Aspergillus fumigatus from 21 clinical isolates were evaluated. Strain detection to evaluate the clinical detection performance of RPA. (6) sensitivity by RPA and PCR simultaneously, The method of RPA and PCR were compared. [results] (1) A primer of Aspergillus fumigatus was obtained, which was located upstream of ITSD1, and 489bp of ITSD2, amplification product. The sequence of the primers was: the upstream primer: 5: 5 GGTCCAACCCCCCCCCCTGTCTATCTATC-3, and the downstream primer: 5TTAGAAAAATATGGGTCGCGGC-3'(2) the genomic DNA extracted from Aspergillus fumigatus by mechanical glass bead cracking method could meet the needs of nucleic acid amplification in purity and quantity, and was simple and convenient. (3) the optimal reaction conditions for the detection of Aspergillus fumigatus by RPA were as follows: primer length was 18-32 BP, amplification product length was 200-700 BP, amplification temperature was 37-42 鈩，

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