Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Targets and Sample Preparation
2.2. RPA and LAMP Reaction in Tube
2.3. Air-Insulated Microfluidic Chip Design
2.4. RPA and LAMP Reactions on Microfluidic Chip
3. Results
3.1. Comparison of Sensitivity and Reproducibility of Basic RPA Followed by Fluorescence LAMP (bRPA-LAMP) with Basic RPA and Fluorescence LAMP in Tube
3.2. Comparison of Sensitivity of bRPA-LAMP with LAMP on the Microfluidic Chip
3.3. Clinical Sensitivity and Specificity of Parallel Detection for Virus on the Same Microfluidic Chip
4. Discussion
- (1)
- Detecting different types of nucleic acid targets on one chip.
- (2)
- Fast detection with high sensitivity and specificity.
- (3)
- Accurate parallel detection of multiple targets with a small volume.
Author Contributions
Funding
Conflicts of Interest
References
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Target | Primer Name | Primer Sequence (5′-3′) |
---|---|---|
SARS-CoV-2 (CoV-2) | CoV-RPA-F | ATACACTAATTCTTTCACACGTGGTGTTTA |
CoV-RPA-R | AGTAGCGTTATTAACAATAAGTAGGGACTGGG | |
CoV-LAMP-F3 | ACACTAATTCTTTCACACGTGGTG | |
CoV-LAMP-B3 | ATTAACAATAAGTAGGGACTGGG | |
CoV-LAMP-FIP | CCAGAGACATGTATAGCATGGAACCCATTCAACTCAGGACTTGTTCT | |
CoV-LAMP-BIP | GAGGTTTGATAACCCTGTCCTACCATCTTCGAATCTAAAGTAGTACC | |
CoV-LAMP-LF | CATTGGAAAAGAAAGGTA | |
CoV-LAMP-LB | TGCTTCCACTGAGAAG | |
Measles Virus (MV) | MV-RPA-F | AGAATAATGAAGAAGGGGGAGACTATTATGA |
MV-RPA-R | CAGCAGCAGCTGTCCTCTGGAACTGGTCCG | |
MV-LAMP-F3 | GGACACCTCTCAAGCATC | |
MV-LAMP-B3 | CAGCAGCTGTCCTCTGGAA | |
MV-LAMP-FIP | CGGCCTGAATCTCTGCCTATGATTGGGAAGGATCCCAACG | |
MV-LAMP-BIP | GTTCTCAAGAAACCCGCTGCCCTGGTCCGTCCATTTGTCA | |
MV-LAMP-LF | GGATTGAGTTCGACATCTGC | |
MV-LAMP-LB | AGCCGACAACTCCAAGGA |
Tp ± SD (min, N = 3) | ||||
---|---|---|---|---|
1E+4 Copies | 1E+3 Copies | 1E+2 Copies | 1E+1 Copies | |
LAMP | 17.8 ± 0.6 | 22.8 ± 0.9 | 2 in 3 positive | NS |
Unpurified RPA product followed by LAMP | 8.4 ± 0.2 | 11.8 ± 0.2 | 35.3 ± 1.4 | 2 in 3 positive |
Purified RPA product followed by LAMP | 8.3 ± 0.1 | 10.4 ± 0.1 | 14.4 ± 0.2 | 21.3 ± 0.5 |
Tp ± SD (min, N = 3) | |||
---|---|---|---|
1E+3 Copies | 1E+2 Copies | 1E+1 Copies | |
bRPA-LAMP | 12.33 ± 0.08 | 17.26 ± 0.07 | 21.02 ± 0.41 |
LAMP | 15.07 ± 0.02 | 21.77 ± 0.46 | 1 in 3 positive |
Targets | Culture | On Microfluidic Chip | Clinical Sensitivity | Clinical Specificity | |
---|---|---|---|---|---|
SARS-CoV-2 | Positive | 17 | 16 | 94.12% | 95.83% |
Negative | 23 | 24 | |||
MV | Positive | 23 | 23 | 100.0% | 100.0% |
Negative | 17 | 17 |
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Huang, Q.; Shan, X.; Cao, R.; Jin, X.; Lin, X.; He, Q.; Zhu, Y.; Fu, R.; Du, W.; Lv, W.; et al. Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus. Micromachines 2021, 12, 1582. https://doi.org/10.3390/mi12121582
Huang Q, Shan X, Cao R, Jin X, Lin X, He Q, Zhu Y, Fu R, Du W, Lv W, et al. Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus. Micromachines. 2021; 12(12):1582. https://doi.org/10.3390/mi12121582
Chicago/Turabian StyleHuang, Qin, Xiaohui Shan, Ranran Cao, Xiangyu Jin, Xue Lin, Qiurong He, Yulei Zhu, Rongxin Fu, Wenli Du, Wenqi Lv, and et al. 2021. "Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus" Micromachines 12, no. 12: 1582. https://doi.org/10.3390/mi12121582