Abstract
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.
Highlights
Multidrug-resistant tuberculosis (MDR-TB) is defined by the resistance of Mycobacterium tuberculosis (Mtb) to at least the two most potent antimicrobials against TB infection, PLOS ONE | DOI:10.1371/journal.pone.0137791 September 10, 2015Detection of MDR-TB by PCR-Nucleic Acid Lateral Flow (NALF)
In order to determine MDR-TB, this study focuses on the positive detection of RIF and INH resistance
85–95% of RIF resistance has been found to naturally accompany INH resistance [17,18,19,20,21,22,23], making RIF resistance a widely used surrogate marker for MDR-TB; there is a 5–15% chance of RIF monoresistance development. 90–95% of RIF resistance confers mutation(s) in the 81-bp hot-spot region of the rpoB gene, called the rifampicin resistancedetermining region (RRDR), with the highest global prevalence being at codons 531, 526 and 516
Summary
Multidrug-resistant tuberculosis (MDR-TB) is defined by the resistance of Mycobacterium tuberculosis (Mtb) to at least the two most potent antimicrobials against TB infection, PLOS ONE | DOI:10.1371/journal.pone.0137791 September 10, 2015. The target Mtb genes for the PCR-NALF test in this study are rpoB, with mutations conferring RIF resistance [9,10], and katG, with mutations conferring INH resistance [11]. Only the primer specific to the mutation type binds to the target, from the multitude of primers, to register RIF resistance This design strategy is practical because a simultaneous occurrence of more than one drug resistance conferring mutation in a single gene is uncommon. All synthesized primers were labeled with specific tags for a rapid and easy detection by NALF antibodies Both rpoB and katG assays follow the same test protocols, including the same thermocycling conditions. All NALF results were compared with the results of agarose gel electrophoresis for laboratory evaluation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
