Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

Table 1 Sensitivity and standard curves of the triplex real-time PCR assay

Number of DNA copies (copies/μL)	triplex real-time PCR Ct Value (mean ± SD)			Uniplex real-time PCR Ct Value (mean ± SD)
Number of DNA copies (copies/μL)	AIV	NDV	DTMUV	AIV	NDV	DTMUV
1 × 10⁷	13.61 ± 0.20	14.50 ± 0.14	14.30 ± 0.33	13.93 ± 0.07	15.01 ± 0.17	14.68 ± 0.41
1 × 10⁶	16.42 ± 0.21	18.00 ± 0.37	17.87 ± 0.09	16.31 ± 0.15	18.22 ± 0.26	18.05 ± 0.04
1 × 10⁵	20.33 ± 0.22	21.39 ± 0.08	21.44 ± 0.09	20.32 ± 0.04	22.26 ± 0.11	21.24 ± 0.16
1 × 10⁴	23.02 ± 0.52	24.41 ± 0.16	24.99 ± 0.05	23.04 ± 0.44	24.71 ± 0.16	24.73 ± 0.43
1 × 10³	26.01 ± 0.37	28.04 ± 0.35	28.30 ± 0.08	25.84 ± 0.20	27.74 ± 0.53	27.78 ± 0.01
1 × 10²	29.55 ± 0.15	30.45 ± 0.36	31.19 ± 0.65	29.30 ± 0.30	30.78 ± 0.47	31.12 ± 0.12
1 × 10¹	32.42 ± 0.68	33.34 ± 0.71	33.29 ± 0.86	32.98 ± 0.34	33.85 ± 0.68	33.38 ± 0.60

Serial 10-fold dilutions from 1 × 10⁷ to 1 × 10¹ copies of pMD-AIV, pMD-NDV, pMD-DTMUV were used as templates to determine the standard curve of the triplex real-time PCR and for uniplex real-time PCR. Each dilution was performed in triplicate for the assays
Ct ≤ 34 was considered positive for AIV, Ct ≤ 35 was considered positive for NDV and DTMUV

ISSN: 1746-6148