Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Vaccine Viruses and Clinical Samples
2.2. Primers and TaqMan Oligoprobes Used for OPV/nOPV Serotype Identification and Quantification
2.3. Extraction of Viral RNA
2.4. qmosRT-PCR Amplification
2.5. Sensitivity of the qmosRT-PCR Assay
2.6. Spike of nOPV2 and Trivalent nOPV in Stool Supernatant
3. Results
3.1. Evaluation of the Specificity and Sensitivity of the Simultaneous Detection and Quantification of Each nOPV Strain in the Presence of High Concentrations of the Two Other Serotypes
3.2. Analysis of Monovalent nOPV2-c1 and Trivalent nOPV Spiked in Stool Supernatant
3.3. Analysis of Stool Samples from nOPV2 Clinical Trials
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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nOPV/OPV Serotype | Oligo Name | Location in nOPV Genome | Sequence 5′-->3′ | Size (nt) | Tm (°C, Basic) | Amplicon Size (bp) |
---|---|---|---|---|---|---|
1 | 2771Sab1F | 2831-2849 | CAGCTTCCACCAAGAATAA | 19 | 43 | 92 |
Sab1-2962R | 2922-2904 | GAAGAACTCCAATTTCCTC | 19 | 47 | ||
Sab1-FAM | 2863-2877 | FAM-ACAGTGTGGAAGATC-NFQ | 15 | |||
2 | 2682TqS2F | 2748-2763 | CCAGAGACGAACGCGA | 16 | 49 | 122 |
2802TqS2R | 2869-2852 | AAACCGAAAACAATCTGC | 18 | 44 | ||
Sab2-VIC | 2772-2787 | VIC-CACGGTTGAGTCATTC-NFQ | 16 | |||
3 | 1411TqS3F | 1479-1497 | GGGAAAATTTTACTCCCAA | 19 | 45 | 69 |
Sab3-1487R | 1547-1527 | ACTGGGCAGAACTCTCTTTTT | 21 | 51 | ||
Sab3-NED | 1510-1524 | NED-AACGCAGTAACATCC-NFQ | 15 |
nOPV Serotypes | Expected Titers * | qmosRT-PCR Result γ | Expected Titers * | qmosRT-PCR Result γ | ||
---|---|---|---|---|---|---|
Ct | log10 CCID50/mL | Ct | log10 CCID50/mL | |||
1 | 3 | 34.96 | 1.35 ± 0.33 | 3 | UD | |
2 | 3 | 26.15 | 3.51 ± 0.08 | 7 | 15.95 | 6.52 ± 0.07 |
3 | 7 | 14.89 | 6.79 ± 0.08 | 7 | 17.06 | 6.22 ± 0.05 |
1 | 3 | 25.55 | 3.81 ± 0.03 | 4 | 32.34 | 2.04 ± 0.09 |
2 | 7 | 14.42 | 6.97 ± 0.06 | 7 | 15.83 | 6.56 ± 0.03 |
3 | 3 | 28.51 | 3.21 ± 0.18 | 7 | 17.04 | 6.23 ± 0.01 |
1 | 7 | 12.05 | 7.34 ± 0.04 | 2 | UD | |
2 | 3 | 25.78 | 3.62 ± 0.03 | 6 | 18.93 | 5.64 ± 0.05 |
3 | 3 | 28.62 | 3.18 ± 0.09 | 6 | 19.95 | 5.46 ± 0.05 |
1 | 7 | 12.04 | 7.35 ± 0.02 | 3 | 29.62 | 2.75 ± 0.01 |
2 | 7 | 14.00 | 7.1 ± 0.03 | 3 | 28.20 | 2.9 ± 0.01 |
3 | 3 | 36.28 | 1.16 ± 0.59 | 6 | 20.07 | 5.43 ± 0.05 |
1 | 7 | 12.08 | 7.33 ± 0.05 | 4 | 23.29 | 4.4 ± 0.11 |
2 | 3 | 26.89 | 3.29 ± 0.06 | 4 | 24.59 | 3.97 ± 0.07 |
3 | 7 | 15.12 | 6.73 ± 0.08 | 6 | 20.28 | 5.37 ± 0.09 |
1 | 3 | UD | 2 | UD | ||
2 | 7 | 14.19 | 7.04 ± 0.02 | 2 | 33.06 | 1.46 ± 0.08 |
3 | 7 | 15.15 | 6.72 ± 0.13 | 6 | 20.19 | 5.4 ± 0.04 |
1 | 2 | UD | ||||
2 | 2 | 31.17 | 2.02 ± 0.07 | |||
3 | 7 | 15.06 | 6.75 ± 0.04 | |||
1 | 2 | 30.83 | 2.43 ± 0.02 | |||
2 | 7 | 14.36 | 6.99 ± 0.02 | |||
3 | 2 | 33.26 | 1.96 ± 0.07 | |||
1 | 7 | 12.08 | 7.33 ± 0.02 | |||
2 | 2 | 28.37 | 2.85 ± 0.09 | |||
3 | 2 | 40.54 | 0.04 ± 0.47 |
nOPV Serotypes | Expected Titer * | qmosRT-PCR Result γ | |
---|---|---|---|
Ct | log10 CCID50/mL | ||
1 | 7 | 12.87 | 7.00 ± 0.02 |
2 | 7 | 16.58 | 6.90 ± 0.03 |
3 | NT | UD | |
1 | 7 | 12.75 | 7.03 ± 0.02 |
2 | NT | UD | |
3 | 7 | 14.79 | 6.91 ± 0.02 |
1 | NT | UD | |
2 | 7 | 17.28 | 6.70 ± 0.01 |
3 | 7 | 14.91 | 6.87 ± 0.02 |
1 | 7 | 12.84 | 7.01 ± 0.01 |
2 | NT | UD | |
3 | NT | UD | |
1 | NT | UD | |
2 | 7 | 17.89 | 6.52 ± 0.02 |
3 | NT | UD | |
1 | NT | UD | |
2 | NT | UD | |
3 | 7 | 15.04 | 6.84 ± 0.03 |
1 | 7 | 12.89 | 6.99 ± 0.03 |
2 | 7 | 16.55 | 6.91 ± 0.05 |
3 | 7 | 14.95 | 6.86 ± 0.03 |
nOPV Serotypes | Expected Titer * | qmosRT-PCR Result | |
---|---|---|---|
Ct | log10 CCID50/mL γ | ||
1 | 7 | 12.98 | 6.97 ± 0.01 |
2 | 7 | 16.63 | 6.89 ± 0.01 |
3 | 1 | 37.04 | 0.55 ± 0.38 |
1 | 7 | 12.55 | 7.09 ± 0.01 |
2 | 1 | 41.79 | −0.25 ± 0.40 |
3 | 7 | 14.67 | 6.94 ± 0.05 |
1 | 1 | 33.43 | 0.83 ± 0.07 |
2 | 7 | 17.27 | 6.70 ± 0.00 |
3 | 7 | 14.98 | 6.85 ± 0.01 |
1 | 6 | 16.44 | 5.93 ± 0.02 |
2 | 6 | 20.48 | 5.78 ± 0.03 |
3 | 1 | 34.04 | 1.40 ± 0.33 |
1 | 6 | 16.24 | 5.99 ± 0.00 |
2 | 1 | 39.16 | 0.51 ± 0.39 |
3 | 6 | 18.14 | 5.94 ± 010 |
1 | 1 | 32.77 | 1.04 ± 0.12 |
2 | 6 | 21.07 | 5.61 ± 0.01 |
3 | 6 | 18.41 | 5.87 ± 0.13 |
1 | 6 | 16.36 | 5.95 ± 0.02 |
2 | 3 | 30.49 | 2.90 ± 0.03 |
3 | 1 | 35.09 | 1.01 ± 0.11 |
1 | 1 | 32.44 | 1.13 ± 0.03 |
2 | 6 | 22.47 | 5.21 ± 0.03 |
3 | 3 | 28.54 | 2.92 ± 012 |
1 | 3 | 25.82 | 3.11 ± 0.04 |
2 | 1 | 37.46 | 0.91 ± 0.17 |
3 | 6 | 18.56 | 5.82 ± 0.09 |
Trivalent nOPV | Monovalent nOPV2-C1 | ||||
---|---|---|---|---|---|
Expected Titers * | nOPV1 Titers γ (Average Ct) | nOPV2-C1 Titers γ (Average Ct) | nOPV3 Titers γ (Average Ct) | Expected Titers * | nOPV2-C1 Titers γ (Average Ct) |
5.52 | 4.86 ± 0.06 (22) | 5.08 ± 0.07(22) | 5.21 ± 0.10 (22) | 5.55 | 4.11 ± 0.09 (26) |
5.05 | 4.32 ± 0.08 (24) | 4.55 ± 0.09 (24) | 4.70 ± 0.14 (24) | 5.08 | 3.60 ± 0.10 (27) |
4.57 | 3.83 ± 0.10 (26) | 4.11 ± 0.14 (25) | 4.21 ± 0.13 (26) | 4.60 | 3.17 ± 0.05 (28) |
4.09 | 3.48 ± 0.11 (27) | 3.81 ± 0.12 (26) | 3.81 ± 0.12 (27) | 4.12 | 2.83 ± 0.04 (29) |
3.61 | 3.06 ± 0.15 (29) | 3.45 ± 0.12 (27) | 3.43 ± 0.15 (28) | 3.64 | 2.54 ± 0.11 (31) |
3.14 | 2.75 ± 0.16 (30) | 3.11 ± 0.15(29) | 3.03 ± 0.18 (30) | 3.17 | 2.30 ± 0.12 (32) |
2.66 | 2.49 ± 0.19 (30) | 2.77 ± 0.19 (30) | 2.57 ± 0.15 (31) | 2.69 | 1.89 ± 0.13 (34) |
2.18 | 1.77 ± 0.18 (33) | 2.03 ± 0.14 (32) | 2.12 ± 0.20 (33) | 2.21 | 1.54 ± 0.24 (34) |
1.71 | 1.51 ± 0.29 (34) | 1.36 ± 0.16(35) | 1.72 ± 0.20 (34) | 1.74 | 1.05 ± 0.31 (35) |
1.23 | 0.45 ± 0.12 (37) | 0.29 ± 0.38 (38) | 1.08 ± 0.28 (36) | 1.26 | 1.09 ± 0.71 (38) |
0.75 | 0.72 ± 0.13 (37) | 0.78 | |||
0.27 | 0.30 |
Sample Codes | Detection Results | Titers (log10 CCID50/mL) | |||||
---|---|---|---|---|---|---|---|
Type 1 | Type 2 | Type 3 | All Types | Type 1 | Type 2 | Type 3 | |
2 | Neg | Pos | Neg | 2 | 2.74 | ||
3 | Neg | Pos | Neg | 2 | 3.43 | ||
4 | Neg | Pos | Pos | 2, 3 | 3.64 | 4.55 | |
5 | Neg | Pos | Neg | 2 | 2.76 | ||
6 | Neg | Pos | Neg | 2 | 2.15 | ||
7 | Neg | Pos | Neg | 2 | 2.15 | ||
8 | Neg | Pos | Neg | 2 | 1.3 | ||
9 | Neg | Pos | Neg | 2 | 2.28 | ||
10 | Neg | Pos | Neg | 2 | 2.71 | ||
11 | Neg | Pos | Neg | 2 | 1.99 | ||
12 | Pos | Pos | Pos | 1, 2, 3 | −0.27 | 5.26 | 0.04 |
13 | Pos | Pos | Pos | 1, 2, 3 | −0.34 | 4.09 | 0.61 |
14 | Neg | Pos | Neg | 2 | 2.46 | ||
15 | Neg | Pos | Neg | 2 | 2.54 | ||
16 | Neg | Pos | Neg | 2 | 5.11 | ||
17 | Neg | Pos | Neg | 2 | 2.83 | ||
18 | Neg | Pos | Neg | 2 | 2.69 | ||
19 | Neg | Pos | Neg | 2 | 0.55 | ||
20 | Pos | Pos | Pos | 1, 2, 3 | 1.85 | 2.47 | 0.11 |
21 | Neg | Pos | Neg | 2 | 2.95 | ||
22 | Neg | Pos | Neg | 2 | 2.25 | ||
23 | Neg | Pos | Neg | 2 | 5.45 | ||
24 | Neg | Pos | Neg | 2 | 3.35 | ||
25 | Neg | Pos | Neg | 2 | 3.43 | ||
26 | Neg | Pos | Neg | 2 | 4.43 | ||
27 | Neg | Pos | Neg | 2 | 4.77 |
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Manukyan, H.; Tritama, E.; Wahid, R.; Anstadt, J.; Konz, J.; Chumakov, K.; Laassri, M. Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples. Vaccines 2023, 11, 1729. https://doi.org/10.3390/vaccines11111729
Manukyan H, Tritama E, Wahid R, Anstadt J, Konz J, Chumakov K, Laassri M. Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples. Vaccines. 2023; 11(11):1729. https://doi.org/10.3390/vaccines11111729
Chicago/Turabian StyleManukyan, Hasmik, Erman Tritama, Rahnuma Wahid, Jennifer Anstadt, John Konz, Konstantin Chumakov, and Majid Laassri. 2023. "Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples" Vaccines 11, no. 11: 1729. https://doi.org/10.3390/vaccines11111729