Internally Controlled, Generic Real-Time PCR for Quantification and Multiplex Real-Time PCR with Serotype-Specific Probes for Serotyping of Dengue Virus Infections
Table 5
Inter-assay variation of generic real-time PCRa.
Run and DENV in vitro RNA copies in PCRa
IC copies in PCRb
Number positive DENV in vitro RNA (%; mean Cq-value: SD)
Number positive IC (%; mean Cq-value: SD)
SD between the 3 runs
Run 1
106
500
4/4 (100%; 26.28; 0.43)
4/4 (100%; 36.21; 1.21)
0.27
105
500
4/4 (100%; 30.40; 0.39)
4/4 (100%; 33.10; 1.37)
0.35
104
500
4/4 (100%; 33.78; 0.38)
4/4 (100%; 32.17; 0.30)
0.22
103
500
2/4 (50%; 36.99; 0.37)
4/4 (100%; 33.64; 0.29)
0.65
102
500
0/4
4/4 (100%; 32.47; 0.20)
—
0
0
0/4
0/4
—
Run 2
106
500
4/4 (100%; 26.71; 0.56)
4/4 (100%; 36.11; 0.84)
0.27
105
500
4/4 (100%; 30.35; 0.19)
4/4 (100%; 32.66; 0.65)
0.35
104
500
4/4 (100%; 34.31; 0.64)
4/4 (100%; 33.32; 0.39)
0.22
103
500
3/4 (75%; 37.08; 0.86)
4/4 (100%; 33.64; 0.92)
0.65
102
500
1/4 (25%; 37.83)
4/4 (100%; 32.73; 1.09)
—
0
0
0/4
0/4
—
Run 3
106
500
4/4 (100%; 26.79; 0.60)
4/4 (100%; 35.13; 1.06)
0.27
105
500
4/4 (100%; 30.98; 0.50)
4/4 (100%; 32.47; 0.10)
0.35
104
500
4/4 (100%; 34.27; 0.78)
4/4 (100%; 33.24; 0.54)
0.22
103
500
1/4 (25%; 38.17)
4/4 (100%; 33.91; 0.67)
0.65
102
500
0/4
4/4 (100%; 32.59; 0.23)
—
0
0
0/4
0/4
—
aThe inter-assay variation was determined with 10-fold dilution series of DENV in vitro RNA in the presence of a fixed amount of IC spiked into lysis buffer containing DENV negative human serum as background and for 4 replicates per each dilution. The variation was determined in three separate runs performed at three different days.
bNumber of copies cDNA/PCR has been calculated as 1/12.5 of the extracted RNA, assuming 100% efficiency in extraction, reverse transcription (RT), and PCR. SD: standard deviation.
