1. Melinda E. Clark, Ph.D.
Division of Consolidated Laboratory Services
World TB Day - 3/24/15
What is a probe? Understanding Nucleic acid Detection tests and results

2. Back to basics. What is a Probe?
Probe (verb): to examine or explore with, or as if with, a probe
We are ‘exploring’ if an isolate or culture has a particular organism in it.
We PROBE for its DNA or RNA

3. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest

4. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest
Probe

5. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest
Probe
Not complementary

6. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest
Probe

7. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest
Probe
Not complementary

8. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
DNA or RNA from organism of interest
Probe
Complementary!!!

9. What is a Molecular Probe?
A small fragment of DNA that is complementary to part of the DNA or RNA of the organism we are searching for.
The probe has a ‘marker’ on it that allows for detection
Bound
Unbound

10. What Can Molecular Probes Recognize?
DNA from organism of interest
Probes to recognize DNA:
GeneXpert MTB/RIF –rpoB gene
RNA from organism of interest
Probes to recognize rRNA:
MTD – amplified rRNA
AccuProbes - rRNA

11. How Does GeneXpert MTB/RIF Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Wild-type MTBC

12. How Does GeneXpert MTB/RIF Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Wild-type MTBC

13. How Does GeneXpert MTB/RIF Work?
5 probes that cover different part of the rpoB gene
Other organisms DNA
No MTBC?

14. How Does GeneXpert MTB/RIF Work?
5 probes that cover different part of the rpoB gene
Point mutation indicative of resistance
rpoB gene
Rif Resistant MTBC

15. How Does GeneXpert MTB/RIF Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Rif Resistant MTBC

16. What Can Molecular Probes Recognize?
DNA from organism of interest
Probes to recognize DNA:
GeneXpert MTB/RIF –rpoB gene
RNA from organism of interest
Probes to recognize rRNA:
MTD – amplified rRNA
AccuProbes - rRNA

17. How Do AccuProbes Work?
Release rRNA
In reality
Add Probe
In reality

18. Now We Know How Probes Work
Let’s look at Sample Differences!

19. What Types of Samples can be used for Probes?
Direct Specimen
Nucleic Acid Amplification Tests (NAAT)
Examples: GeneXpert MTB-RIF, GenProbes MTD, Roche COBAS TaqMan MTB Test, Hain FluoroType MTB
Culture Specimen
Isolates or mixed
Examples: Gen-Probes Accuprobe

20. What Do We Learn from a NAAT?
What Do We Learn From the Culture Probe?
What Do We Learn from a NAAT?
Remember: Primary Specimens!!
Remember: Culture based specimen!!
The test tells you:
If the organisms’ DNA is present
It DOES NOT tell you
If it is viable
If treatment is working
If there are other organisms present
The test tells you
The identity of the organism growing
The original specimen has viable organisms
It CANNOT tell you
If there are other organisms present

21. Tips on Determining what was Tested

25. Terminology to look for:
NAAT/ Direct specimen probes:
Amplified by direct test
Culture Probe:
AFB organism ID by DNA probe
AFB seen in broth culture. Mycobacterial probes for MTBC have been added.
AFB Detected in culture. See AFB organism ID by DNA probe (then lists tuberculcosis complex, avium complex, kansasii, gordonae under “Test name”.

26. Not to be Confused with:

27. Other Types of “Probes”
Sequencing!
16S rDNA sequencing to ID organisms
PCR of specific genes
IS6110- insertion element found only in MTBC

28. A Brief Moment on Ribosomal RNA terminology
16S rDNA sequencing to ID organisms
rDNA = DNA that encodes for ribosomal RNA
the rRNA gene
We sequence the rDNA
HENCE we sequence the rRNA gene
16S rRNA = ssu rRNA = 16S ribosomal RNA

29. Ribosomes
Prokaryotes have 70S ribosomes: 30S small subunit and 50S large subunit
Focus on the 30S
The 30S subunit from Thermus thermophilus
Protein = blue
Single RNA chain = orange
30S = 16S RNA and 21 proteins
The 16S RNA is responsible for recognizing 5’ end of mRNA to initiate translation!

30. 16s sequencing of rDNA (rRNA gene)
Amplified

31. 16s sequencing of rDNA (rRNA gene)
Complementary strand built – but gets truncated.

32. 16s sequencing of rDNA (rRNA gene)
Complementary strand built – but gets truncated.

33. 16s sequencing of rDNA (rRNA gene)
Complementary strand built – but gets truncated.

34. 16s sequencing of rDNA (rRNA gene)
Complementary strand built – but gets truncated.

35. DNA Fragments Separated Based on Size
Color of Each nucleotide detected and the sequence is deciphered.

36. Sequence Determined!
Color of Each nucleotide detected and the sequence is deciphered.

37. Sequence is Compared to Known Sequences
Our Sequence
M. kansasii
M. tuberculosis
M. avium

38. Sequence is Compared to Known Sequences
Our Sequence
Therefore our sequence = M. tuberculosis
M. tuberculosis
M. avium
M. kansasii

39. Terminology to look for
ID by 16S rDNA
ID by 16S rRNA
ID by 16s rRNA gene sequencing
ID by small ribosomal subunit sequencing
ID by ssu rRNA
If you see ID by PCR
Not sequencing, but detection of a specific gene

40. Further Questions? Melinda E. Clark, Ph.D. Elaine McCaffery, MT (ASCP)
Phone: x223
Elaine McCaffery, MT (ASCP)
Phone: x210