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Table 2 Evaluation of Filtration methods to improve sensitivity and specificity of rt-qPCR assays

From: A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers

Dilutions (3 × replicates)

Hydrolysis probe assay

SYBR Green assay

  

Crossing point (SD), Quant value (SD)

Filtered

5.0 × 105

23.38 (0.03),

540 000 (9 000)

20.95 (0.68)

850 000 (400 000)

 

1.0 × 105

25.36 (0.16)

160 000 (16 000)

24.20 (0.44)

81 000 (26 000)

 

2.0 × 104

28.33 (0.17)

27 000 (3000)

26.14 (0.31)

20 000 (4000)

 

1.0 × 103

33.74 (0.44)

1000 (300)

32.65 (0.49)

200 (70)

 

5.0 × 102

34.64 (0.45)

600 (160)

33.29 (1.24)

160 (130)

 

2.5 × 102

37.40 (n/a)

110 (n/a)

37.02 (1.05)a

10 (10)

 

1.0 ×02

Not detected

 

False Positive

20 (10)

Unfiltered

5.0 × 105

24.53 (0.62)

300 000 (94 000)

22.83 (0.46)

200 000 (70 000)

 

1.0 × 105

25.46 (0.35)

156 000 (33 000)

24.50 (0.11)

64 000 (4800)

 

2.0 × 104

29.15 (0.05)

17 000 (500)

29.85 (0.44)

1500 (500)

 

1.0 × 103

33.81 (0.38)

1000 (220)

32.98 (0.16)

160 (20)

 

5.0 × 102

35.27 (1.63)

550 (450)

36.38 (0.23)a

15 (3)

 

2.5 × 102

38.35 (1.06)

75 (50)

35.63 (0.73)a

30 (10)

 

1.0 ×02

Not detected

 

False Positive

10 (2)

  1. a Indicates nonspecific melting peaks of 79°C obtained by melting curve analysis of replicates tested.
  2. n/a = Not Applicable
  3. SD = Standard Deviation
  4. ND = Not Detected