Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Clinical Isolates
2.2. Primers
2.3. Preparation of PAC and IAC Templates
2.4. Preparation of DNA Templates from Clinical Isolates
2.5. Development of Heptaplex PCR Assay
2.6. Thermostabilization and Stability Evaluation of the Heptaplex PCR Assay
2.7. Analytical Sensitivity and Specificity Evaluation of the Heptaplex PCR Assay
3. Results
3.1. Development of the Heptaplex PCR Assay
3.2. Thermostabilization and Stability Evaluation of the Heptaplex PCR Assay
3.3. Sensitivity and Specificity Evaluation of the Heptaplex PCR on the Reference Strains and Clinical Isolates
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Primers | Sequence (5′ → 3′) | Target Amplicon | Size (bp) |
---|---|---|---|
IAC_F 1AC_R | F: AAC TTA TCC CCA ATC GCG CA R: GCC CTT TCT TCT CAA GCG GT | H. pylori (IAC) | 105 |
1_F 2_R | F: ATT TCT CCG GCG TCA AGT GT R: CTC AAC ATC GTC GCA AAG GC | K. pneumoniae | 202 |
3_F 4_R | F: CGC AAA CTG TTG GCC ACT AT R: CTC GCC ATC ATG ATT CAA GT | S. aureus | 293 |
5_F 6_R | F: TTG ACC CAT CAG GGA GAA AG R: CTT GAT GCC ACT TAG CCA AC | S. pneumoniae | 349 |
7_F 8_R | F: GAT GGA AAT GCT GAA ATT CG R: GGA CGC TCT TTA CCA TAG GA | P. aeruginosa | 444 |
9_F 10_R | F: TGG AGG ATC CGT ACG AGA AG R: TTG ACA GTG GAC ACC TTG GA | M. tuberculosis complex | 505 |
11_F 12_R | F: GCG AAA GTC CAA GCC TCT CT R: TCA CCG TAA GAT ACT GTG CCT | H. influenzae | 582 |
Organism | Reference Strains, n | Clinical Isolates, n | Total Test, n | Positive Test, n (%) | Negative Test, n (%) | |
---|---|---|---|---|---|---|
1. | K. pneumoniae | 2 | 19 | 21 | 21 (100) | 0 (0) |
2. | S. aureus | 2 | 16 | 18 | 18 (100) | 0 (0) |
3. | S. pneumoniae | 3 | 17 | 20 | 20 (100) | 0 (0) |
4. | P. aeruginosa | 2 | 21 | 23 | 23 (100) | 0 (0) |
5. | M. tuberculosis | 2 | 16 | 18 | 18 (100) | 0 (0) |
6. | H. influenzae | 2 | 17 | 19 | 19 (100) | 0 (0) |
7. | A. hydrophila | 1 | 0 | 1 | 0 (0) | 1 (100) |
8. | A. baumannii | 1 | 2 | 3 | 0 (0) | 3 (100) |
9. | Acinetobacter spp. | 0 | 3 | 3 | 0 (0) | 3 (100) |
10. | A. xylosoxidans | 0 | 2 | 2 | 0 (0) | 2 (100) |
11. | B. cereus | 1 | 0 | 1 | 0 (0) | 1 (100) |
12 | B. subtilis | 1 | 0 | 1 | 0 (0) | 1 (100) |
13. | B. pseudomallei | 0 | 1 | 1 | 0 (0) | 1 (100) |
14. | C. freundii | 0 | 2 | 2 | 0 (0) | 2 (100) |
15. | Enterobacter species | 0 | 2 | 2 | 0 (0) | 2 (100) |
16. | E. aerogenes | 1 | 0 | 1 | 0 (0) | 1 (100) |
17. | E. cloacae | 1 | 0 | 1 | 0 (0) | 1 (100) |
18. | E. coli | 1 | 6 | 7 | 0 (0) | 7 (100) |
19. | E. coli O157 | 1 | 0 | 1 | 0 (0) | 1 (100) |
20. | Klebsiella spp. | 0 | 1 | 1 | 0 (0) | 1 (100) |
21. | L. monocytogenes | 1 | 0 | 1 | 0 (0) | 1 (100) |
22. | M. catarrhalis | 0 | 2 | 2 | 0 (0) | 2 (100) |
23. | N. gonorrhoeae | 1 | 0 | 1 | 0 (0) | 1 (100) |
24. | N. meningitidis | 1 | 0 | 1 | 0 (0) | 1 (100) |
25. | P. mirabilis | 1 | 0 | 1 | 0 (0) | 1 (100) |
26. | R. gilardii | 0 | 1 | 1 | 0 (0) | 1 (100) |
27. | S. epidermis | 1 | 0 | 1 | 0 (0) | 1 (100) |
28. | S. marcescens | 0 | 1 | 1 | 0 (0) | 1 (100) |
29. | S. mutans | 1 | 0 | 1 | 0 (0) | 1 (100) |
30. | S. pyogenes | 1 | 0 | 1 | 0 (0) | 1 (100) |
31. | S. sanguinis | 1 | 0 | 1 | 0 (0) | 1 (100) |
32. | S. viridians | 1 | 2 | 3 | 0 (0) | 3 (100) |
33. | Serratia marcescens | 0 | 1 | 1 | 0 (0) | 1 (100) |
34. | Streptococcus group A | 0 | 5 | 5 | 0 (0) | 5 (100) |
35. | Streptococcus group B | 0 | 4 | 4 | 0 (0) | 4 (100) |
36. | Streptococcus group C | 0 | 2 | 2 | 0 (0) | 2 (100) |
37. | Streptococcus group G | 0 | 2 | 2 | 0 (0) | 2 (100) |
Evaluation of the developed multiplex PCR assay for RTIs: | ||||||
Total test, N | : | 175 | ||||
Total positive | : | 119/119 | (100%) | |||
Total negative | : | 57/57 | (100%) | |||
Test accuracy | : | 175/175 | (100%) |
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Nik Zuraina, N.M.N.; Goni, M.D.; Amalina, K.N.; Hasan, H.; Mohamad, S.; Suraiya, S. Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens. Diagnostics 2021, 11, 753. https://doi.org/10.3390/diagnostics11050753
Nik Zuraina NMN, Goni MD, Amalina KN, Hasan H, Mohamad S, Suraiya S. Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens. Diagnostics. 2021; 11(5):753. https://doi.org/10.3390/diagnostics11050753
Chicago/Turabian StyleNik Zuraina, Nik Mohd Noor, Mohammed Dauda Goni, Khazani Nur Amalina, Habsah Hasan, Suharni Mohamad, and Siti Suraiya. 2021. "Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens" Diagnostics 11, no. 5: 753. https://doi.org/10.3390/diagnostics11050753