Analysis And Interpretation Of Results - INGENASA INgene q IBV SYBR Instrucciones De Uso

6. Reading the fluorescence takes place during the elongation step (marked in the table)*, and the channels through
which the fluorescence data are collected are detailed in table :
Passive reference

9. ANALYSIS AND INTERPRETATION OF RESULTS

Each problem sample analyzed yields data regarding the fluorescence originated from channel SYBR, both amplification
(pathogen detection) and melting curve analysis. Automatic threshold analysis (threshold and baseline) is recommended.
If this option is not viable, the analysis can be carried out manually following the thermocycler instructions.
The assay will be considered as valid when the C+ have a Ct value within the range 25+4 and the C- Ct>40 in the SYBR
Possible results are summarised in the table:
SYBR channel: pathogen detection
A positive result during amplification implies a typical fluorescence curve with a Ct value <35, with a specific Tm similar to
that of positive amplification control (A1).
Samples with Ct´s comprised between 35 and 40 should be regarded as doubtful. In that case we recommend:
• Repeating the analysis.
• Sequencing the amplified product.
Case Problem sample
1 +
2 -
3
4
Case 1. Both the problem sample and the positive PCR control exhibit a positive result, whereas the negative extraction
and PCR controls yield a negative result.
Positive result: the sample contains IBV.
Case 2. The positive PCR control exhibits a positive result, whereas the problem sample and the negative extraction and
PCR controls yield a negative result.
Negative result: the sample does not contain any IBV.
Case 3.The negative extraction control yields a positive result.
Result invalidated or null. Go back to extraction step.
Case 4. The negative extraction control exhibits a negative result, whereas the negative PCR control yields a positive
result. Result invalidated or null. Go back to amplification step.
INSTRUCTIONS – ENGLISH
Reporter
IBV SYBR
ROX
Table: Results validation
Negative control
Positive PCR control
of extraction
-
-
+
-
Emission wavelength
520 nm
610 nm
channel
Negative
PCR control
+ -
+ -
+
Quencher
None
Result of problem sample
+
-
Null
Null
(Versión 1410-14) – PAG.11.