New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

Figures & data

Figure 1. Comparative analysis of Ct values generated by distinct concentrations of recombinant plasmid containing the 83 bp fragment from the NS5 region of the 17DD yellow fever virus (YFV) diluted in negative human serum (YFV standard curve) and in serum containing together Mumps virus, Dengue 1, 2 and 3 and Measles (YFV standard curve + viral pool).

Table 1. Specificity analysis of the Yellow Fever Virus RT-qPCR

Sample	Threshold Cycle (Ct)
Yellow fever virus 10^6 copies/reaction	19.31
Yellow fever virus 10^5 copies/reaction	22.76
Yellow fever virus 10^4 copies/reaction	25.78
Yellow fever virus 10^3 copies/reaction	29.60
Yellow fever virus 10^2 copies/reaction	34.54
Dengue virus 1 10^5 copies/reaction	ND
Dengue virus 2 10^5 copies/reaction	ND
Dengue virus 3 10^5 copies/reaction	ND
Japanese encephalitis virus 10^5 copies/reaction	ND
Negative controls (human serum of healthy individual and non-template controls - NTC)	ND

ND, Not Detected.

Table 2. Linearity between the number of plasmid copies/reaction and Ct values to estimate the limits of detection and quantification for the Yellow Fever Virus RT-qPCR

Plasmid Copy Number (copies/reaction)	Ct mean	Standard Deviation	Number of positive replicates/Number of replicates
10^7	16.93	0.16	8/8
10^6	20.59	0.06	8/8
10^5	24.08	0.11	8/8
10^4	27.50	0.13	8/8
10^3	31.10	0.41	8/8
10^2	34.17	0.49	8/8 QL
50	34.88	18.07	5/8
25	35.53	18.40	5/8 DL
12.5	36.13	18.70	3/8
6.2	ND	-	0/8

Ct = Threshold Cycle; ND = Not Detected; QL = Quantification Limit; DL = Detection Limit.

Figure 2. Reportable amplification range to evaluate the performance of the TaqMan YFV RT-qPCR. The standard curve parameters were: slope = - 3.68; intercept = 45.08; coefficient of determination (r²) = 0.99 and amplification efficiency (E) = 86.2 %. Each point of the curve was tested in 8 replicates in the same run.

Figure 3. Precision evaluation of the YFV RT-qPCR. (A) Reconstituted serological samples spiked with purified 17DD-YFV: high viral load - 10⁴ copies/reaction, average viral load - 10³ copies/reaction and low viral load - 10² copies/reaction. Each viral concentration was assayed in 8 replicates. The experiments were performed by 2 different operators and repeated in 4 separate assays; 2 experiments were conducted on the same day and the other 2 were carried out on different days (standard deviations are indicated). (B) Clinical samples from 17DD-YFV vaccinated individuals represented in 3 distinct concentrations: high viral load - 10³ copies/reaction, average load - 10² copies/reaction and low viral load - 50 copies/reaction. Each viral concentration was assayed in 10 replicates. The assays were conducted by 2 different operators and repeated in 3 independent tests; 2 experiments were performed on the same day by different operators and one test was conducted on a different day by one operator (standard deviations are indicated).

Figure 4. In vitro samples of 17DD-YFV propagated in serum-free medium from 2 bioreactor vases analyzed by RT-qPCR for the NS5 viral region and for the EXO IPC (exogenous control), in separate reactions on the same plate. The experiments were performed during the first 4 d of viral propagation. Dotted lines indicate the results for EXO IPC, showing minimal Ct variation between samples.

Table 3. Evaluation of the human RNase P gene as an endogenous internal amplification control in the TaqMan multiplex YFV RT-qPCR. Ct values for both RNAse P and NS5 region from yellow fever virus are indicated, as well as the standard deviation (SD). The samples were assayed in 8 replicates each

Sample	Mean Ct (Yellow Fever Virus)	± SD	Mean Ct (human RNase P)	±SD
Clinical sample (Low viral load)	36.40	0.96	31.30	0.16
Clinical sample (Average viral load)	35.84	0.83	31.41	0.02
Clinical sample (High viral load)	34.95	0.83	31.93	0.02
Serological panel (Low viral load)	35.94	0.23	30.22	0.11
Serological panel (High viral load)	30.96	0.15	30.43	0.30
Negative serum	ND	30.86

Ct = Threshold Cycle; ND = Not Detected.

* Pool of samples constituted by human sera from 17DD yellow fever virus vaccinated individuals.

** Serological panel generated by spiking non-infected serum from a healthy individual (Negative Quality Control Serum) with purified 17DD yellow fever virus.

Figure 5. Correlation between the YFV RT-qPCR method (copies/mL) and virus titration (PFU/mL), from 38 analyzed samples (clinical samples from YFV vaccinated individuals and in vitro propagated virus), indicated by the equation: Log₁₀ PFU/mL = [0.974 × Log₁₀ copies/mL] – 2.807.