Molecular diagnosis of tuberculosis

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 available.

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 be present

• 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 template. – 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.

Logaritmic multiplication

Transcription Mediated Amplification (TMA)

• 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 and DNA.

• TMA has several other differences in comparison to PCR and LCR:

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 15–30 minutes.

(v) The RNA strands created in iv are able to base pair to the 2nd primer (step 9).

Subsequently, the reverse transcriptase catalyzes the formation of a cDNA strand by extending the 3′ end of the 2nd primer, and again an RNA/DNA hybrid duplex results (step 10)

(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 detection.

NAA tests for direct detection

• 1- Amplicor Mycobacterium tuberculosis Test (Amplicor; Roche Diagnostic Systems,Inc., Branchburg, NJ).

• 2- Enhanced Mycobacterium tuberculosis Direct Test (E-MTD; Gen- Probe, San Diego, CA)

AmplicorDNA is amplified with genus-specific primers formulated on the basis of the 16S rRNA gene.

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 amplicons

The conjugate then reacts with peroxide and TMB in dimethylformamide to form a color complex

results are measured with a photometer

Amplicor

• 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 –91.9%,

• the specificity is 99.6 –99.8%,

E-MTD

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

E-MTD• 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 smear-negative specimens.

• The overall sensitivity (compared with culture) for respiratory specimens is 90.9 –95.2%,

• the specificity is 98.8–100%,

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