重组酶介导等温核酸扩增技术快速检测烟曲霉菌
发布时间：2018-08-25 12:05
【摘要】：【目的】以烟曲霉内转录间隔区(ITSD)为靶序列,筛选烟曲霉菌特异性的引物对,应用重组酶介导的等温核酸扩增(RPA)技术建立快速检测烟曲霉的新方法并对该检测方法进行方法学优化。【方法】(1)以烟曲霉菌的易变区内转录间隔区为靶序列,先用Primer-5软件在不同区间设计三条上游引物和三条下游引物,交叉配对组成不同引物对,然后将引物逐一进行Primer-blast,筛选烟曲霉的特异性引物。(2)应用机械性玻璃珠打击裂解法抽提获得烟曲霉标准菌株的DNA。(3)通过优化RPA检测烟曲霉的引物长度、产物长度、扩增时间及扩增温度等检测条件,建立检测烟曲霉的新方法。(4)通过交叉扩增实验及检测不同浓度烟曲霉DNA样品来评估RPA检测烟曲霉菌的敏感性和特异性。(5)通过对21株临床分离的人致病性曲霉菌菌株的检测来评估RPA技术的临床检测性能。(6)通过RPA与PCR同时进行敏感性、特异性、临床分离曲霉菌检测试验,对RPA与PCR技术进行方法学比较。【结果】(1)筛选得到烟曲霉特异性的引物为上游位于ITSD1、下游位于ITSD2、扩增产物为489bp的引物。引物序列为:上游引物:5'-GGTCCAACCTCCCACCCGTGTCTATC-3',下游引物:5'-TTAGAAAAATAAAGTTGGGTGTCGGC-3'(2)通过机械性玻璃珠打击裂解法抽提获得的烟曲霉基因组DNA在纯度和的量上能够满足核酸扩增需要,且简便、快速。(3)RPA技术检测烟曲霉菌的优化反应条件为:引物长度范围为18-32bp;扩增产物长度范围为200-700bp;扩增温度为37-42℃;扩增时间为15-40min。(4)RPA特异性和敏感性:不同曲霉菌种交叉扩增实验显示,RPA只在烟曲霉菌扩增中有明显的特异性条带,而对黄曲霉、土曲霉、黑曲霉、构巢曲霉、中国红酵母均无扩增。在以琼脂糖凝胶电泳为检测平台时,RPA可以检测到最低浓度为100pg/μl的烟曲霉DNA样品。(5)21株临床分离的人致病性曲霉菌检测实验中,RPA对18株烟曲霉分离菌株的扩增均为阳性,而3株黄曲霉扩增均为阴性。(6)通过对RPA和PCR检测烟曲霉方法学的比较,RPA和PCR一样具有很高的敏感性和很强的特异性。【结论】本研究通过Primer-Blast筛选针对烟曲霉ITS1-ITS2靶DNA序列的特异性引物。在优化反应条件下,应用此特异性引物建立的RPA检测烟曲霉方法可以在较低的恒温条件下对烟曲霉菌DNA进行有效扩增,具有简便、快速、成本低的特点,而且与PCR技术具有相同的敏感性和和特异性,适合用于烟曲霉菌的快速检测和鉴定。
[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，
本文编号：2202829
[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，
本文编号：2202829
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