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为了建立一种能同时检测1类整合子、4类超级整合子和1型插入序列共同区的多重PCR检测方法,根据Gen Bank中1类整合子整合酶基因int1、4类超级整合子整合酶基因intSXT和1型插入序列共同区转座酶基因ISCR1的序列,应用Primer Plex 2.61设计3对特异性引物,通过改变引物浓度和退火温度对多重PCR体系进行了优化。此后,评价了所建立多重PCR体系的特异性和灵敏度,并应用于222株海水养殖源弧菌中3种整合子相关元件的检测。结果显示,设计的3对引物能分别单独且特异性地扩增出569 bp、430 bp和651 bp的目的条带;当int1、intSXT和ISCR13对引物(10 pmol/μL)的体积依次为0.3μL、0.6μL和0.6μL,退火温度为50℃时,多重PCR体系能特异性地扩增出3个条带清晰、区分度良好的目的条带,方法的灵敏度达到0.02 ng/μL弧菌基因组模板DNA;利用所建立的方法对222株临床分离弧菌进行3种整合子元件检测得到的结果与文献报道的单引物检测结果一致。从上述研究结果可以看出,该多重PCR方法能快速准确地检测Int1、SXT和ISCR1,适用于整合子相关元件流行监测及整合子相关的耐药性研究。
In order to establish a multiplex PCR method that can simultaneously detect class 1 integrons, 4 types of super integron and 1 type insertion region common region, according to the gen Bank type 1 integrase gene int1, 4 super integron integrase The sequence of the transposase gene ISCR1 shared by intSXT and type 1 insertions was designed. Primer Plex 2.61 was used to design three pairs of specific primers. The multiplex PCR system was optimized by changing primer concentration and annealing temperature. Since then, the specificity and sensitivity of the established multiplex PCR system were evaluated and applied to the detection of three integron-related elements in 222 strains of marine cultured vibrio. The results showed that the designed 3 pairs of primers could separately and specifically amplify the target bands of 569 bp, 430 bp and 651 bp. When the volumes of the primers (10 pmol / μL) for int1, intSXT and ISCR13 were 0.3 μL, 0.6μL, 0.6μL and annealing temperature of 50 ℃, the multiplex PCR system can specifically amplify three bands with clear and well-differentiated bands with sensitivity of 0.02 ng / μL Vibrio genomic The results of three kinds of integron elements detection of 222 clinical isolates of Vibrio parahaemolyticus were consistent with those of the single primer reported in the literature. From the above results, it can be seen that the multiplex PCR method can rapidly and accurately detect Int1, SXT and ISCR1, and is suitable for the epidemiological surveillance of integron-related elements and the integron-related drug resistance research.