Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viral and Bacterial Strains
2.2. Clinical Specimens
2.3. Primer
Organism | GenBank Accession No. | Target Gene | Oligonucleotide Sequence of Primer (5’–3’) | Length (bp) | Position (5’–3’) |
---|---|---|---|---|---|
M. tuberculosis | CP009480.1 | IS986 | F- CTTAACATTTAACTTCTATAACAC-12C-CGTGAGGGCATCGAGGTGGCR-biotin-GCGTAGGCGTCGGTGACAAA | 245 | 889650–889894 |
C . neoformans | L38588.1 | URA | F- CACTTAATTCATTCTAAATCTATC-12C-TGTCCTAACCAGTGCGACAGCGATGR-biotin-GTACTTCCTGACCTCTTGCAGCTCC | 360 | 390–749 |
S. pneumoniae | AF467249.1 | lytA | F- ATTAAACAACTCTTAACTACACAA-12C-CGCAATCTAGCAGATGAAGCAGGTT R-biotin-AAGGGTCAACGTGGTCTGAGTGGTT | 124 | 328–451 |
HSV-1 | X14112.1 | Gene 42 | F- TACATTCAACACTCTTAAATCAAA-12C-GCCGTTGAGCTAGCCAGCGA R-biotin-GTGCTGGTGCTGGACGACAC | 257 | 93557–93813 |
HSV-2 | Z86099.2 | TK | F- ACTACTTATTCTCAAACTCTAATA-12C-GTAAGCGCGGGCCAAAGGATR-biotin-TCAAACACGGAAGCCCGAAC | 234 | 46635–46868 |
2.4. Plasmid Preparation
2.5. DNA Extraction and PCR Amplification
2.6. PCR Product Identification and Luminex Assay
2.7. Determination of Cutoff Values and Data Analysis
3. Results
3.1. Specificity of Primers
3.2. Determination of Amplification Conditions for Five-Plex PCR
3.3. Analytical Specificity of PCR-Luminex Assay
3.4. Analytical Sensitivity of PCR-Luminex Assay
Organism | Plasmid Quantity (Copies) | cMFI |
---|---|---|
M. t uberculosis | 108 | 16.95 ± 0.45 |
106 | 10.06 ± 0.22 | |
104 | 7.49 ± 0.30 | |
102 | 6.79 ± 0.20 | |
101 | 0.93 ± 0.07 | |
HSV-1 | 102 | 11.41 ± 0.72 |
50 | 6.56 ± 0.20 | |
10 | 5.30 ± 0.31 | |
5 | 2.30 ± 0.12 | |
1 | 2.45 ± 0.15 | |
HSV-2 | 108 | 17.72 ± 0.59 |
106 | 7.14 ± 0.38 | |
104 | 6.19 ± 0.14 | |
102 | 3.49 ± 0.07 | |
101 | 2.25 ± 0.02 | |
C. neoformans | 108 | 34.80 ± 0.21 |
106 | 31.86 ± 0.18 | |
104 | 28.29 ± 0.45 | |
102 | 9.62 ± 0.12 | |
101 | 1.23 ± 0.10 | |
S. pneumoniae | 108 | 8.78 ± 0.47 |
106 | 6.15 ± 0.36 | |
104 | 4.94 ± 0.29 | |
102 | 3.77 ± 0.22 | |
101 | 2.18 ± 0.14 |
3.5. Assessment of PCR-Luminex Performance Using CSF Specimens
PCR-Luminex | Smear Positive | Smear Negative | Sensitivity | Specificity | Kappa |
---|---|---|---|---|---|
(+) | 49 | 1 | 90.7% | 99.1% | 0.9153 |
(−) | 5 | 108 |
PCR-Luminex | MGG Positive | MGG Negative | Sensitivity | Specificity | Kappa |
---|---|---|---|---|---|
(+) | 23 | 4 | 92% | 97.1% | 0.8628 |
(−) | 2 | 134 |
PCR-Luminex | ELISA Positive | ELISA Negative | Sensitivity | Specificity | Kappa | |
---|---|---|---|---|---|---|
HSV-1 | (+) | 21 | 8 | 80.8% | 94.2% | 0.7158 |
(−) | 5 | 129 | ||||
HSV-2 | (+) | 1 | 1 | 100% | 99% | 0.6639 |
(−) | 0 | 161 |
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Appendix
References
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Zhou, L.; Wu, R.; Shi, X.; Feng, D.; Feng, G.; Yang, Y.; Dai, W.; Bian, T.; Liu, T.; He, Y.; et al. Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology. Int. J. Environ. Res. Public Health 2016, 13, 193. https://doi.org/10.3390/ijerph13020193
Zhou L, Wu R, Shi X, Feng D, Feng G, Yang Y, Dai W, Bian T, Liu T, He Y, et al. Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology. International Journal of Environmental Research and Public Health. 2016; 13(2):193. https://doi.org/10.3390/ijerph13020193
Chicago/Turabian StyleZhou, Linfu, Rui Wu, Xiaodan Shi, Dongyun Feng, Guodong Feng, Yining Yang, Wen Dai, Ting Bian, Tingting Liu, Ying He, and et al. 2016. "Simultaneous Detection of Five Pathogens from Cerebrospinal Fluid Specimens Using Luminex Technology" International Journal of Environmental Research and Public Health 13, no. 2: 193. https://doi.org/10.3390/ijerph13020193