A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood

BMC Microbiology

Table 3 Comparison of sensitivity of the nested multiplex PCR and multiplex PCR methods in real time

Microbe species	Order of magnitude [CFU/ml]						Detection limit of the multiplex qPCR	Order of magnitude [CFU/ml]						Detection limit of the nested multiplex qPCR [CFU/ml]	*RFU level
	10⁵	10⁴	10³	10²	10¹	10⁰	[CFU/ml]	10⁵	10⁴	10³	10²	10¹	10⁰
	+	+	+	∅	∅	∅		+	+	+	+	+	∅		100
*A. fumigatus*	+	+					3.7 × 10 ³ ± 2.4 × 10 ³	+	+	+	+			3.7 × 10 ¹ ± 2.7 × 10 ²
(filamentous fungi)	+	+					3.2 × 10³ CFU/reaction	+	+	+	+			1.2 CFU/reaction
	+	+					**C_T (31.2-38.5)	+	+	+	+			C_T (29.1-32.2)
	+	+						+	+	+	+
	+	+	+		∅	∅		+	+	+	+		∅		50
*C. albicans*	+	+	+	+			9.9 × 10 ² ± 3.4 × 10 ³	+	+	+	+	+		8.5 × 10 ¹ ± 3.6 × 10 ²
(yeast)	+	+	+	+			9.5 × 10¹ CFU/reaction	+	+	+	+	+		0.24 CFU/reaction
	+	+	+				C_T (33.3-37.2)	+	+	+	+	+		C_T (29.7-32.1)
	+	+	+					+	+	+	+	+
	+	+	+	∅	∅	∅		+	+	+	+	+	+		100
*S. aureus*	+	+	+				4.5 × 10 ³ ± 2.5 × 10 ³	+	+	+	+	+		1.1 × 10 ¹ ± 3.7 × 10 ²
(Gram positive bacteria)	+	+	+				5.1 × 10² CFU/reaction	+	+	+	+	+		0.78 CFU/reaction
	+	+	+				C_T (34.0-36.7)	+	+	+	+	+		C_T (25.2-28.0)
	+	+						+	+	+	+	+
	+	+		∅	∅	∅		+	+	+	+	+	+		30
*E. coli*	+	+	+				5.4 × 10 ³ ± 2.5 × 10 ²	+	+	+	+	+		1.3 × 10 ¹ ± 3.7 × 10 ²
(Gram negative bacteria)	+	+	+				6.1 × 10² CFU/reaction	+	+	+	+	+		0.73 CFU/reaction
	+	+	+				C_T (30.5-33.2)	+	+	+	+	+		C_T (24.4-27.2)
	+	+	+					+	+	+	+	+

“+”- the number of positive amplification results conducted in the five repetitions; “∅” – lack of amplification signal.
*RFU (relative fluorescence unit) signifies the value of the designated baseline.
**C_T value, i.e. the consecutive reaction cycle number in which the linear increase of the product cut the established baseline RFU level.

ISSN: 1471-2180