Pre PCR specimen processing/extraction Flashcards
Describe the specimen types acceptable for various tests in a molecular pathology laboratory.
Endocervical swabs (females), urethral swabs (M), nasal swabs (MRSA), EDTA, EDTA with gel separator (pear top tubes), spinal fluids and lesion swaps (herpes), formalin fixed paraffin embedded tissue (for cancer markers)
List the components in the master mix solution used for PCR
DNA polymerase- enzyme needed to replicate the target sequence primers to initiate replication Magnesium buffers to maintain pH Deoxyribonucleotide and probes
Explain why using an internal control is mandatory for each molecular test performed.
The internal control is a oligonucleotide sequence that is different from that of the target but binds the same primers. it’s product is detected using a different probe to detect the target sequence. if the internal control is not amplified= invalid test. causes include the presence of a PCR inhibitor, denaturation of the polymerase, hybridization failure, or errors in the detection system.
Identify the specific drug that inhibits PCR
Heparin
summarize common practices in the molecular laboratory to prevent contamination
-aerosol barrier pipette tips, preparation of reagents in a dead air box or biological cabinet, separate area for performing, 10% bleach with 95% alcohol destroy amplicons, different lab coat, gloves/washing hands, DNA AWAY
discuss the importance of specialized pipette tips used in the molecular laboratory
- cotton plugged tips- prevents aerosol contamination of samples
- tall tips- prevents contamination from pipette holder
- DNAse and RNAse free plastic pipettes
explain the guidelines for storing and shipping specimens for the molecular laboratory
- most specimens (swabs and spinal fluids) at 4C
- Plasma sample at -80C
- Thin prep vials at RT
- transported on dry ice then placed in a refrigerator before testing
