Direct Detection of Mycobacteria from Specimens
• Nucleic acid amplification (NAA) methods allow for
detection of mycobacterial DNA or RNA directly
from the specimens before the culture results are
What is Nucleic Acid Amplification (NAA) ?
• Exponential amplification of a specific sequence
of nucleic acid
• - NAA helps to increase the sensitivity of the
assay especially when only a few organisms may
• Two most common types
- Polymerase Chain Reaction (PCR)
- Transcription Mediated Amplification (TMA)
• - Amplified nucleic acid product (amplicon)
detected by specific DNA probe or analyzed by
DNA sequence analysis
PCR - Polymerase Chain Reaction
• PCR is an in vitro technique for the amplification of a region of DNA
which lies between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which
are complementary to the outer regions of known sequence.
• The oligonucleotides serve as primers for DNA polymerase and the
denatured strands of the large DNA fragment serves as the
– This results in the synthesis of new DNA strands which are
complementary to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the
oligonucleotide primers), whereas the 3' ends are potentially
ambiguous in length.
• A target nucleic acid amplification method that uses RNA
transcription (RNA polymerase) and DNA synthesis
(reverse transcriptase) to produce RNA amplicon from a
target nucleic acid. TMA can be used to target both RNA
• TMA has several other differences in comparison to PCR
1. TMA is isothermal. A water bath or heat block is used
instead of a thermal cycler
2. TMA produces RNA amplicon rather than DNA amplicon.
Since RNA is more labile in the laboratory
environment than DNA, this helps reduce the possibility
of carry-over contamination.
3. TMA produces 100-1000 copies per , This results in a 10
billion fold increase of DNA (or RNA) copies within about
(v) The RNA strands
created in iv are able
to base pair to the
2nd primer (step 9).
formation of a cDNA
strand by extending
the 3′ end of the 2nd
primer, and again an
duplex results (step
(i) A promoter-primer binds to the RNA target and
is extended via the DNA polymerase activity of
the reverse transcriptase (RT)
The product of this reaction is an RNA/DNA hybrid
duplex, with the new DNA strand being
complementary to the RNA target (step 3).
(ii) RNase H activity of the reverse transcriptase
specifically digests the RNA strand of the RNA/DNA
hybrid, leaving only the cDNA (step 4).
(iii) the 2nd base pairs to its complementary
sequence on the newly created DNA strand, and
the reverse transcriptase catalyzes the synthesis of
another new DNA strand using the cDNA as a
template (step 5)
The product of this reaction is a DNA intermediate
that contains the RNA polymerase promoter
sequence in double-stranded form (step 6).
(iv) The RNA polymerase now
recognizes the promoter
sequence on the DNA
intermediate and transcribes
multiple copies of the RNA
amplicon (steps 7 and 8).
The RNA amplicon strands are
opposite in polarity from the
original RNA target and contain a
region complementary to the
probe used for amplicon
NAA tests for direct detection
• 1- Amplicor Mycobacterium tuberculosis Test
(Amplicor; Roche Diagnostic Systems,Inc.,
• 2- Enhanced Mycobacterium tuberculosis Direct Test
(E-MTD; Gen- Probe, San Diego, CA)
DNA is amplified with genus-specific
primers formulated on the basis of the 16S
amplicons are denatured to form single strands
and added to a microtiter plate containing a
bound, M.tuberculosis complex-specific probe.
avidin-horseradish peroxidase conjugate
then binds to the bound, biotin-labeled
The conjugate then reacts with peroxide
and TMB in dimethylformamide to form a
results are measured with a photometer
• False-positive results produced by carryover
contamination are prevented by the incorporation of
dUTP coupled with uracil--glycosylase restriction.
• the Amplicor results are available in ;6.5 h
• The overall sensitivity of the Amplicor test (compared
with culture) for respiratory specimens is 79.4 –
• the specificity is 99.6 –99.8%,
RNA amplicons are detected with an acridinium ester-labeled
DNA probe in a solution hybridization assay.
The new transcripts serve as templates for reverse transcription
and further amplification
then transcribed by DNA-directed RNA polymerase to produce
more rRNA molecules
The initial RNA strand is degraded, and a second primer binds to the cDNA and
is extended, leading to the formation of double-stranded cDNA
Reverse transcriptase is then used to copy rRNA to a cDNA-RNA hybrid.
rRNA is released from the target cells by sonication,and a
promoter-primer binds to the rRNA target
• The E-MTD test can be completed in 3.5 h.
• The E-MTD test has been reported to perform
well with both AFB smear-positive and
• The overall sensitivity (compared with
culture) for respiratory specimens is 90.9 –
• the specificity is 98.8–100%,