Molecular microbiology

Question 1

What are the steps involved in processing specimens for NAATs?

Answer 1

• Nucleic acid extraction (lysis, purification, recovery)
• Amplification
• Detection
• Interpretation

Question 2

What are the components of the PCR?

Answer 2

• DNA
• forward and reverse primers
• deoxynucleotide triphosphates
• heat stable DNA polymerase
• buffers and MgCl2

Question 3

What is reverse transcription PCR?

Answer 3

RT-PCR used the enzyme reverse transcriptase to convert RNA to complementary DNA (cDNA) which can be used as a template for PCR

Question 4

What is multiplex PCR and what is required for the reaction?

Answer 4

Used to detect multiple target in a single reaction. This type of PCR requires primers for each target sequence

Question 5

What is real time PCR and what is the advantage of this variation?

Answer 5

Real time PCR uses fluorescence to detect DNA during amplification. The fluorescence is proportional to amount of DNA made. There is no electrophoresis required and thus there are faster results.

Question 6

What is DNA sequencing and what is it used for?

Answer 6

DNA sequencing is used to identify unique genetic signatures. Applications: ID of organisms, predict susceptibility to anti microbials, and outbreak investigations

Question 7

Describe 16s rDNA PCR and sequencing

Answer 7

Targets the conserved and divergent regions of 16s ribosomal DNA found in prokaryotes. The PCR targets the highly conserved regions of the DNA to detect bacteria. The divergent regions of the DNA are then sequenced to ID the bacteria. The sequences can then be compared to a database.