Journal of Shandong University (Health Sciences) ›› 2020, Vol. 58 ›› Issue (10): 127-133.doi: 10.6040/j.issn.1671-7554.0.2020.0923

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Optimization, validation and analysis of a 2019-nCoV nucleic acid detection kit

ZHOU Yunying1, ZHANG Tong2, ZHAO Qianqian1, WANG Haiyan2, WANG Yunshan3   

  1. 1. Medical Research &
    Laboratory Diagnostic Center, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250013, Shandong, China;
    2. Shandong ACV Biotech Limited Company, Jinan 250110, Shandong, China;
    3. Research Center of Basic Medicine, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan 250013, Shandong, China
  • Published:2020-10-08

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Abstract: Objective To optimize the sensitivity and specificity of a 2019-nCoV nucleic acid detection kit, so as to improve the positive detection rate and provide guidance for clinical use by comparison with different kits. Methods From January to March 2020, 88 confirmed and 572 negative specimens of 2019-nCoV were recruited from 6 2019-nCOV nucleic acid testing centers including Jinan Central Hospital Affiliated to Shandong University. By sequence analysis of the whole genome of 2019-nCoV, a more sensitive primer and probe were designed to optimize the buffer ratio and amplification process. The sensitivity was evaluated with cross-reactive experiment and endogenous substances interference test. The optimized kit and 3 other commercial kits were compared with gradient dilution tests. Results The optimized ORF1ab and N gene had higher sensitivity to the RNA targets of 2019-nCoV. When the concentration of virus was 1, the virus could be detected, and the amplification curve was better than the commercial kits. The sensitivity of ORF1ab was increased by 2 times(1∶10 vs 1∶5)compared with WHO or other self-designed kits, and the sensitivity of N gene was increased by 8 times(1∶80 vs 1∶10)and 2 times(1∶80 vs 1∶40)compared with CDC and WHO kits respectively. The results of cross-reactive test and endogenous substances interference test showed that the accuracy of nasopharyngeal swabs was 100%, there was no cross-reaction with other pathogens with similar infection symptoms, and the accuracy and specificity of the kit were 100%. Conclusion The optimized 2019-nCoV nucleic acid detection kit has higher sensitivity and specificity, which is helpful to solve the false negative nucleic acid detection and increase the positive rate, and is significant to formulate discharge criteria of low viral load patients.

Key words: Novel coronavirus, Low viral load, False negative, Sensitivity, Specificity

CLC Number: 

  • R574
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