Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

Abstract

e Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources. With one-third of the world's population infected, tuberculosis (TB) is a major public health threat worldwide (1). The two most important drugs used to treat TB are isoniazid (INH) and rifampin (RIF). Inadequate or inappropriate therapy, together with long and costly treatment courses, often result in the emergence of drug resistance (2). Multidrug-resistant (MDR) Mycobacterium tuberculosis strains-defined as bacillary resistance to at least INH and RIF-have spread rapidly and become a global health emergency (3). Identification of these strains by drug susceptibility testing (DST) allows optimization of therapeutic efficacy and improved treatment outcomes while minimizing transmission of drug-resistant strains. However, phenotypic DST involving subculture is costly, it is too slow to guide optimal management of MDR-TB, and it lacks high-throughput capability. Commercial systems for rapid molecular identification of RIF resistance are available but expensive.RIF resistance in M. tuberculosis is associated with amino acid changes in the ␤-subunit of RNA polymerase, encoded by rpoB. The majority (90 to 95%) of mutations responsible for resistance are located in the 81-bp RIF resistance determining region (RRDR) of rpoB (4-6), and ca. 60 to 70% are found within two codons, 531 and 526. The fact that most mutations known to confer resistance are localized to a specific region facilitates the development of molecular methods for their detection. Sequencing and hybridization-based assays have been commonly used and can define the precise mutations involved (4, 7). Hybridization-based assays also provide a rapid...

Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

“…The combination of padlock-probe circularization and amplification through RCA has proven useful for sensitive and specific detection of nucleic-acid sequences from pathogens (Zhang et al, 1998 ). RCA has gained much popularity in the identification of viral (Haible et al, 2006 ) and bacterial pathogens (Chen et al, 2014 ). Despite its limited use in mycological diagnosis, several studies have demonstrated the applicability of RCA for the molecular identification of medically relevant fungi such as Candida (Zhou et al, 2008 ), Aspergillus (Zhou et al, 2008 ), Trichophyton (Kong et al, 2008 ), Fonsecaea (Najafzadeh et al, 2011 ), Madurella (Ahmed et al, 2014 ), and Mucorales (Dolatabadi et al, 2014 ).…”

Rapid Identification of Black Grain Eumycetoma Causative Agents Using Rolling Circle Amplification

“…The circular structured molecule then amplifies with DNA polymerase that has strand displacement and progressive DNA synthesis activity resulting in series of repeats of the original circular template [11] , [12] . The technique has been proven to be rapid, specific and low-cost for molecular identification of viruses, bacteria, and fungi [13] , [14] , [15] , [16] . It has been applied for identification of a rare black grain mycetoma species Exophiala jeanselmei [17] .…”

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

“…This approach can detect gene mutations associated with drug resistance by using the oligonucleotide probe, which is characteristic of mutation discrimination capability. Such probes used in the previously reported assays include the molecular beacon [ 13 , 14 ], the TaqMan minor groove binder (MGB) probe [ 15 ], the sloppy molecular beacon[ 10 ], the dual-labeled probe[ 16 , 17 ], and the padlock probe[ 18 ] and others. However, none of these probes exhibit rigid specificity to discriminate single-base mutations in the RRDR of M .…”

“…The sloppy molecular beacon-based and dual-labelled probe-based approaches need additional Tm analyses after the amplification[ 19 ]. Padlock probes were used in the rolling circle amplifications, which required complex procedure [ 18 ]. In addition to above probes, LNA probe is another kind of probe that has been widely used in real-time PCR to quantitatively detect pathogens [ 20 , 21 ].…”