Transcript Interpreting DNA results: differentiating between naat and DNA

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
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
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
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
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
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
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
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!!!
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
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
How Does GeneXpert MTB/RIF
Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Wild-type
MTBC
How Does GeneXpert MTB/RIF
Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Wild-type
MTBC
How Does GeneXpert MTB/RIF
Work?
5 probes that cover different part of the rpoB gene
Other organisms DNA
No
MTBC?
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
How Does GeneXpert MTB/RIF
Work?
5 probes that cover different part of the rpoB gene
rpoB gene
Rif Resistant
MTBC
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
How Do AccuProbes Work?
Release
rRNA
In reality
Add Probe
In reality
Now We Know How Probes Work
Let’s look at Sample
Differences!
What Types of Samples can be used
for Probes?
 Direct Specimen


Nucleic Acid Amplification
Tests (NAAT)
Examples: GeneXpert MTBRIF, GenProbes MTD, Roche
COBAS TaqMan MTB Test,
Hain FluoroType MTB
 Culture Specimen


Isolates or mixed
Examples: Gen-Probes
Accuprobe
What Do We Learn
from a NAAT?
What Do We Learn
From the Culture
Probe?
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
Tips on Determining what was
Tested
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”.
Not to be Confused with:
Other Types of “Probes”
 Sequencing!
 16S rDNA sequencing to ID organisms
 PCR of specific genes
 IS6110- insertion element found only in MTBC
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
Ribosomes
 Prokaryotes have 70S
ribosomes: 30S small
subunit and 50S large
subunit
 Focus on the 30S
Protein = blue
Single RNA
chain =
orange
The 30S subunit from
Thermus thermophilus
http://en.wikipedia.org/wiki/File:10_small_subunit.gif
30S = 16S RNA and 21 proteins
http://nar.oxfordjournal
s.org/content/suppl/20
01/12/18/30.1.183.DC
1/Tthe_B_SSU_var_1
50dpi.gif
The 16S RNA is responsible for
recognizing 5’ end of mRNA to
initiate translation!
16s sequencing of rDNA (rRNA
gene)
rDNA
Amplified
16s sequencing of rDNA (rRNA
gene)
DNA (rRNA gene)
Complementary
strand built – but
gets truncated.
16s sequencing of rDNA (rRNA
gene)
DNA (rRNA gene)
Complementary
strand built – but
gets truncated.
16s sequencing of rDNA (rRNA
gene)
DNA (rRNA gene)
Complementary
strand built – but
gets truncated.
16s sequencing of rDNA (rRNA
gene)
DNA (rRNA gene)
Complementary
strand built – but
gets truncated.
DNA Fragments Separated Based on Size
Color of Each
nucleotide detected
and the sequence is
deciphered.
Sequence Determined!
Color of Each
nucleotide detected
and the sequence is
deciphered.
Sequence is Compared to
Known Sequences
Our Sequence
M. kansasii
M. tuberculosis
M. avium
Sequence is Compared to
Known Sequences
Our Sequence
Therefore our sequence =
M. tuberculosis
M. tuberculosis
M. kansasii
M. avium
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
Further Questions?
 Melinda E. Clark, Ph.D.
Phone: 804-648-4480 x223
Email: [email protected]
 Elaine McCaffery, MT (ASCP)
Phone: 804-648-4480 x210
Email: [email protected]