Detection method details
Name: |
QL-ELE-cat-F2/cat-R2 |
Description: |
Qualitative conventional PCR method for detection of the full-length chloramphenicol marker that is frequently harboured by GMM in food enzyme preparations (Fraiture et al. 2020).
In detail, the potential presence of the chloramphenicol marker is first screened using a real-time PCR method (see Related Methods QL-ELE-cat-F/R). In case a positive real-time PCR signal is obtained, the presence of the full-length chloramphenicol marker is then evaluated by a nested-PCR consisting of the related method QL-ELE-cat-F1/cat-R1 and this method. |
Comment: |
Target is the chloramphenicol acetyl-transferase (cat) gene conferring resistance to chloramphenicol (CmR).
For the nested PCR the majority of the chloramphenicol marker sequence is amplified using the primer pair cat-F1/cat-R1 (see Related Methods). A 1:100 dilution of the generated PCR product is then submitted to a second PCR reaction using the primer pair cat-F2/cat-R2 (this method) in order to increase the specific amplicon yield. |
Validation: |
unknown |
Standardisation: |
none |
Related Methods: |
QL-ELE-cat-F/R, QL-ELE-cat-F1/R1 |
Type: |
element-specific |
Target DNA element: |
V-chloramphenicol acetyl transferase
|
-
Oligonucleotides:
Forward Primer
Name:
cat-F2Sequence:
CCAACAAACGACTTTTAGTATAACCSize:
25
Reverse Primer
Name:
cat-R2Sequence:
TCCTGCATGATAACCATCACSize:
20
-
Amplicon:
Sequence:
CCAACAAACGACTTTTAGTATAACCACAGAAATTGATATTAGTGTTTTATACCGAAACATAAAACAAGAAGGATATAAATTTTACCGTGCATTTATTTTCTTAGTGACAAGGGTGATAAACTCAAATACAGCTTTTAGAACTGGTTACAATAGCGACGGAGAGTTAGGTTATTGGGATAAGTTAGAGCCACTTTATACAATTTTTGATGGTGTATCTAAAACATTCTCTGGTATTTGGACTCCTGTAAAGAATGACTTCAAAGAGTTTTATGATTTATACCTTTCTGATGTAGAGAAATATAATGGTTCGGGGAAATTGTTTCCCAAAACACCTATACCTGAAAATGCTTTTTCTCTTTCTATTATTCCATGGACTTCATTTACTGGGTTTAACTTAAATATCAATAATAATAGTAATTACCTTCTACCCATTATTACAGCAGGAAAATTCATTAATAAAGGTAATTCAATATATTTACCGCTATCTTTACAGGTACATCATTCTGTTTGTGATGGTTATCATGCAGGASize:
529