Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay.

Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection

Example of possible NALF outcomes.

<p>Example of possible NALF outcomes.</p

Bacteria and plasmid; source and function in template preparation.

<p>Bacteria and plasmid; source and function in template preparation.</p

NALF and agarose gel electrophoresis results for the <i>rpoB</i> assay.

<p><b>(A)</b> The use of SM primers demonstrate an effective template distinction with the appearance of NALF T1 and T2 for all MT templates to indicate RIF resistant Mtb isolates, which relates to the inner and outer DNA bands on agarose gel for the MT templates. As for WT templates, the assays correctly resulted in the appearance of NALF T2 only, to indicate RIF susceptible Mtb isolates, which corresponds to PCR product size 314 bp on agarose gel.</p

<i>RpoB</i> primer design strategy.

<p><i>RpoB</i> primer designs for codon 526 (mutations Y and R) have been demonstrated above as examples. SM (single mutation) primers are designed with a single base mismatch at the 3’-terminus to complement MT templates. DM (double mutation) primers, which have also been designed to complement MT templates, carry an added mismatch at the third position from the 3’-terminus. The red colored bases indicate an intentional mismatch and the blue colored bases indicate a complementary base to the SM and DM primers.</p

PCR Amplification Products.

<p><b>(A)</b> Multiplex PCR amplification products for the <i>rpoB</i> assay and <b>(B)</b> Semi-nested PCR amplification products for the <i>katG</i> assay, on agarose gel electrophoresis.</p

The least limit of detection (LOD);

<p>For both the <i>rpoB</i> and the <i>katG</i> assays, the NALF results present the LOD as 104 DNA copies (0.1 ng) per PCR reaction, whereas the gel electrophoresis results present the LOD as105 DNA copies (1 ng) per PCR reaction. Below these DNA concentrations, the NALF test lines or the agarose gel DNA bands are not observable. The LOD results for <i>rpoB</i> codon 531 have been shown above to represent the results for other <i>rpoB</i> codons.</p

<i>RpoB</i> assay in multiplex PCR.

<p>NALF results for multiplex PCR presented with the correct appearance of T1 and T2 for MT templates to register RIF resistance Mtb isolates, and the appearance of just T2 for WT template to register RIF susceptible Mtb isolates.</p

Distribution of MDR-TB determining mutations.

<p>A broad compilation of MDR-TB global epidemiological statistics represents the estimated percentage distribution of MDR-TB determining mutations. The compilation demonstrates that 85–95% of RIF resistance is accompanied by INH resistance to determine MDR-TB (purple). Five to fifteen percent of RIF resistance (brown crest) and 5–25% of INH resistance (blue crest) are monoresistance occurrences. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref009" target="_blank">9</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref021" target="_blank">21</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref037" target="_blank">37</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref039" target="_blank">39</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137791#pone.0137791.ref052" target="_blank">52</a>].</p

NALF and agarose gel electrophoresis results for the <i>katG</i> assay.

<p>NALF; the appearance of T1 and T2 for <i>katG</i> MT template indicates a positive detection of INH resistant Mtb isolate, whereas the appearance of T2 (only) for WT template indicates a positive detection of an INH susceptible Mtb isolate. Correspondingly, for gel electrophoresis, two bands are generated for MT template (630 bp, equivalent to NALF T2, and 335 bp to NALF T1), and a single band for WT template (630 bp, equivalent to NALF T2).</p