Curr Microbiol. 2021 Oct;78(10):3656-3666. doi: 10.1007/s00284-021-02621-7. Epub 2021 Aug 02.

Design and Evaluation of Multiplex One-Step Reverse Transcription PCR-Dipstick Chromatography Method for the Analysis of Seven Respiratory Pathogens.

Current microbiology

Li Luo, Qianming Chen, Sheng Qin, Qiang Luo, Zhenjie Liu, Qiong Li, Shuilan Zheng, Xianzhang Huang, Peifeng Ke, Xiangsheng Yang, Hui Xiao, Ning Xu

PMID: 34338833 PMCID: PMC8326646 DOI: 10.1007/s00284-021-02621-7

Abstract

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)-dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA-DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin-streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR-dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR-dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.

© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

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MeSH terms

• COVID-19*
• Chromatography
• Humans
• Multiplex Polymerase Chain Reaction
• Reverse Transcription*
• SARS-CoV-2
• Sensitivity and Specificity

Publication Types

• Journal Article

Grant support

• YN2019QL05/Chinese Medicine Science and Technology Research Project of Guangdong Provincial Hospital of Chinese Medicine
• 202002020038/guangzhou science and technology plan project
• 202103000025/guangzhou science and technology plan project