Background
Methods
Virus stock and clinical samples
Nucleic acid extraction
Design of primers and probes
Primer Name | Sequence (5′ → 3′) | Nucleotide | Length | Matching of primer and probe | ||
---|---|---|---|---|---|---|
0–2 bp | 3–4 bp | ≥5 bp | ||||
RF1 | ATTTTTACGTGCCCAGACAAGAGCCAATGTTCAG | 4986–5019/ | 34 bp | 96.4%(53/55) | 0 (0/55) | 3.6%(2/55) |
RF2 | CAAGAGCCAATGTTCAGATGGATGAGATTCTCAG | 5003–5036/ | 34 bp | 96.4%(53/55) | 0 (0/55) | 3.6%(2/55) |
RR1 | TCAGATGGGTTGGCGTCACTCGACGCCATCTTC | 5087–5119/ | 33 bp | 96.4%(53/55) | 3.6%(2/55) | 0 (0/55) |
RR2 | GACCCATCAGATGGGTTGGCGTCACTCGACGCC | 5093–5125/ | 33 bp | 92.7%(51/55) | 7.3%(4/55) | 0 (0/55) |
RR3 | TTGGCTGTGGACCCATCAGATGGGTTGGCGTC | 5103–5134/ | 32 bp | 96.4%(53/55) | 3.6%(2/55) | 0 (0/55) |
RR4 | ACGAGGTTGGCTGTGGACCCATCAGATGGGTTGGC | 5106–5140/ | 35 bp | 96.4%(53/55) | 3.6%(2/55) | 0 (0/55) |
RR5 | ACCAGGGGCTTGTACAAAATTGTTTCTAATCCAG | 5234–5267/ | 34 bp | 94.5%(52/55) | 5.5%(3/55) | 0 (0/55) |
RR6 | TTCTAGGGGATACTGTAAACTCTCCACCAG | 5263–5292/ | 30 bp | 100%(55/55) | 0 (0/55) | 0 (0/55) |
RR7 | TGTTTCTAATCCAGGGGTCAATTACATTTTGT | 5216–5247/ | 32 bp | 98.2%(54/55) | 1.8%(1/55) | 0 (0/55) |
RR8 | AGCCATAACCTCATTGTTGACCTCTGGGACGAG | 5136–5168/ | 33 bp | 98.2%(54/55) | 1.8%(1/55) | 0 (0/55) |
RR9 | TGGCCAAATGGGAAAGGTAGGGGTTCAGATCAG | 5332–5364/ | 33 bp | 49.1%(27/55) | 45.4%(25/55) | 5.5%(3/55) |
RR10 | ATTCTGGCCAAATGAGAAAGGTAGGGATTCAG | 5337–5368/ | 32 bp | 47.3%(26/55) | 47.3%(26/55) | 5.5%(3/55) |
RR11 | TATTTCACCTGGAGCGTTTCTAGGGGATACTG | 5278–5309/ | 32 bp | 96.4%(53/55) | 3.6%(2/55) | 0 (0/55) |
ROPa | TCAGACCTGAGCACGTGGGAGGGCGATCGCAAFHQGGCTCCCAGTTTTGT | 5033–5082/ | 50 bp | 96.4%(53/55) | 3.6%(2/55) | 0 (0/55) |
Preparation of viral standards
RT-qPCR assay
RT-RAA assay
Evaluation of specificity and sensitivity
Evaluation of the RT-RAA assay using clinical samples
Statistical analysis
Results
Development and screening of RT-RAA primer and probe sets
Specificity of RT-RAA
Sensitivity of RT-RAA
Copies/reaction | Times of positive sample tested by two different assays for NoV (n = 8) | |
---|---|---|
RT-qPCR | RT-RAA | |
2.67 × 107 | 8 | 8 |
2.67 × 106 | 8 | 8 |
2.67 × 105 | 8 | 8 |
2.67 × 104 | 8 | 8 |
2.67 × 103 | 8 | 7 |
2.67 × 102 | 8 | 6 |
2.67 × 101 | 6 | 1 |
2.67 × 100 | 2 | 0 |
Comparison of the RT-RAA and RT-qPCR assays with clinical samples
RT-qPCR | RT-RAA | Total | Agreement | Kappa | p-value of kappa | |
---|---|---|---|---|---|---|
Positive | Negative | |||||
Positive | 16 | 2 | 18 | 88.9% | 0.894 | < 0.001 |
Negative | 0 | 20 | 20 | |||
Total | 16 | 22 | 38 |