Multiplex Nested Solid Phase PCR-Array Chip for Simultaneous Detection of Highly Pathogenic Microorganisms
Abstract Infectious diseases caused by highly pathogenic microorganisms are the main factors threatening human health and causing social panic. To simultaneously detect bacteria and viruses in a single test, a PCR-Array chip based on solid phase PCR (SP-PCR) was developed, in which an 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. Due to lack of real sample and consideration of biological safety, plasmid 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 technology. The primer fixation efficiency and solid-phase RT-PCR reaction system were optimized with four positive samples. The results showed that the detection limit of the multiplex solid-phase RT-PCR was 10 copy μL−1. This method avoided mutual interference among the primers by isolation them on space and improved the detection thoughput, showing high sensitivity and specificity in detection of bacteria and viruses.