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

ZHU Can-Can ^1,2, ,
CUI Jun-Sheng ¹ ,
HU An-Zhong ¹ ,
YANG Ke ¹ ,
ZHAO Jun ^1,2 ,
LIU Yong ¹ ,
DENG Guo-Qing ¹ ,
ZHU Ling ¹

1.
Institute of Applied Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Anhui Provincial Engineering Technology Research Center for Biomedical Optical Instrument, Anhui Provincial Engineering Laboratory for Medical Optical Diagnosis & Treatment Technology and Instrument, Hefei 230031, China;
2.
University of Science and Technology of China, Hefei 230026, China

Received Date: 16 May 2019
Revised Date: 26 August 2019

Fund Project: This work was supported by the Innovation Project, Chinese Academy of Sciences (No. KGFZD-135-18-019), the Program of the Science and Technology Service Network Initiative and the Key Research and Development Program of Anhui Province of China (Nos. 1804c04020335, 1804a09020051).

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.

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

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