多重巢式固相PCR-Array芯片用于高致病性病原微生物并行检测

Multiplex Nested Solid Phase PCR-Array Chip for Simultaneous Detection of Highly Pathogenic Microorganisms

提出了一种多重巢式固相PCR的方法,以微流控芯片为载体,采用紫外交联方式将寡核苷酸序列固定在芯片上,构建了阵列式固相PCR-阵列(PCR-Array)芯片,用于细菌和病毒类高致病性病原微生物的并行检测。选择新疆出血热病毒、埃博拉病毒、炭疽杆菌和布鲁氏菌4种代表性高致病性病原微生物为研究对象,通过基因工程手段构建了包含有炭疽杆菌和布鲁氏菌特异性基因片段的重组质粒载体,利用病毒包装技术构建了包含有两种病毒特异性基因片段的逆转录病毒颗粒,完成了4种高致病性病原微生物阳性标准品的制备,并以此作为实验样本,探究了多重巢式固相PCR-Array芯片的制备方法和扩增体系。结果表明,在优化的实验条件下,多重巢式固相PCR-Array芯片检测4种高致病性病原微生物的检出限达10 copy/μL以下量级。本方法灵敏度高,特异性好,同时,引物空间上的隔离避免了相互干扰,提高了检测通量,在多种病原微生物快速并行检测方面具有良好的应用前景。

To simultaneously detect bacteria and viruses in a single test, a PCR-array chip based on solid phase PCR(SP-PCR) was developed. Ultraviolet cross-linking method was used to fix oligonucleotide sequence on the grass to develop a PCR array chip for detection of Crimea-Congo hemorrhagic fever virus (CCHFV), Ebola virus, Bacillus anthracis and Brucella multiplex. Owing to the lack of real sample and consideration of biological safety, the vectors of Bacillus anthracis and Brucella were constructed by genetic engineering. Meanwhile, to simulate the reverse transcription process of CCHFV virus and Ebola virus, virus-like particle (VLP) containing two specific viral fragments was constructed by virus packaging technique. The primer fixation efficiency and solid-phase RT-PCR reaction system were optimized with four positive samples. The results showed that the optimum time of cross-linking was 9 min, the optimum concentration was 100 μmol/L , and the minimum detection limit of the multiplex solid-phase RT-PCR was under 10 copy/μL. This method with high sensitivity and specificity avoided mutual interference among the primers by isolation them on space and improved the detection flux. This PCR-Array chip would be widely employed for the rapid parallel detection of bacteria and viruses.