Two Color Multiplexing and Typing of Human Papillomavirus Types 16, 18 and 45 on LightCycier 1m
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Melting curves
Segment 1
Segment 2
Segment 3
Target temperature [0C)
95
48
70
Incubation time [s I
10
180
0
Temperature transition rate [20°C/s]
20
5
0.2
Acquisition mode
None
None
Step
• Color compensation was used to correct signal bleed through of channels 1
and 2 into channel 3.
The melting curve profile for each product was analyzed using the "Melting Curve Analysis
Analysis" format of the LightCycler Data Analysis (LCDA) software. The "Melting
Curve Analysis" format uses unique algorithms that take the negative derivative
of the product melting curve generating in turn melting peaks. Once in this program, there are certain parameters that must be set for the detection of HPV high
risk types.
• The Melting Curve calculation needs to be set as "polynomial with background
subtraction" in both channels 2 and 3 to compensate for the non-specific effects
of temperature on fluorescence.
• The background subtraction cursors (2 green and 2 blue vertical lines) in F2
need to be placed at the following temperatures (OC): 51.00,52.00,64.00 and
66.00. The cursors in F3 need to be set at the following temperatures (0C):
53.00,54.00,66.00 and 68.00.
• "oC to Average" which sets the temperature interval used to calculate the derivative data should be placed at a value of 8.0.
Results
Two color multiplexing and analysis of HPV 16, HPV 18 and HPV 45 was accomplished on the LightCycler instrument. HPV 18 and HPV 45 were detected by the
HPV 18/45 LC-Red 640 sensor probe and identified in channel 2 based on melting temperature. The HPV 18 product had a melting temperature of about 59.57°C
while the HPV 45 product had a melting temperature of 56.06°C (see figures 2).
The 4 C difference in melting temperature was used to differentiate these two
closely related HPV high risk types making the analysis fast, uncomplicated and
reliable. The HPV 16 product was detected by the LC-Red 705 sensor probe and
seen in channel 3. The melting temperature of the HPV 16 product appeared to be
approximately 60.66°C (see figure 2). In summary, the HPV 18 and HPV 45 products can be differentiated by melting temperature while HPV 18 and HPV 16
products can be distinguished by color. The HPV 16 and HPV 45 products can be
distinguished both by melting temperature and color.
Detection of HPV 16,
HPV 18 and HPV 45
• Melting Curve Analysis
Parameter
Value
Cycles
Type
Melting curves
Segment 1
Segment 2
Segment 3
Target temperature [0C)
95
48
70
Incubation time [s I
10
180
0
Temperature transition rate [20°C/s]
20
5
0.2
Acquisition mode
None
None
Step
• Color compensation was used to correct signal bleed through of channels 1
and 2 into channel 3.
The melting curve profile for each product was analyzed using the "Melting Curve Analysis
Analysis" format of the LightCycler Data Analysis (LCDA) software. The "Melting
Curve Analysis" format uses unique algorithms that take the negative derivative
of the product melting curve generating in turn melting peaks. Once in this program, there are certain parameters that must be set for the detection of HPV high
risk types.
• The Melting Curve calculation needs to be set as "polynomial with background
subtraction" in both channels 2 and 3 to compensate for the non-specific effects
of temperature on fluorescence.
• The background subtraction cursors (2 green and 2 blue vertical lines) in F2
need to be placed at the following temperatures (OC): 51.00,52.00,64.00 and
66.00. The cursors in F3 need to be set at the following temperatures (0C):
53.00,54.00,66.00 and 68.00.
• "oC to Average" which sets the temperature interval used to calculate the derivative data should be placed at a value of 8.0.
Results
Two color multiplexing and analysis of HPV 16, HPV 18 and HPV 45 was accomplished on the LightCycler instrument. HPV 18 and HPV 45 were detected by the
HPV 18/45 LC-Red 640 sensor probe and identified in channel 2 based on melting temperature. The HPV 18 product had a melting temperature of about 59.57°C
while the HPV 45 product had a melting temperature of 56.06°C (see figures 2).
The 4 C difference in melting temperature was used to differentiate these two
closely related HPV high risk types making the analysis fast, uncomplicated and
reliable. The HPV 16 product was detected by the LC-Red 705 sensor probe and
seen in channel 3. The melting temperature of the HPV 16 product appeared to be
approximately 60.66°C (see figure 2). In summary, the HPV 18 and HPV 45 products can be differentiated by melting temperature while HPV 18 and HPV 16
products can be distinguished by color. The HPV 16 and HPV 45 products can be
distinguished both by melting temperature and color.
Detection of HPV 16,
HPV 18 and HPV 45