Rapid Detection of rpoB Gene Mutations in Rifampin-Resistant Mycobacterium tuberculosis Isolates in Shanghai by Using the Amplification Refractory Mutation System

Abstract

Rapid detection of drug resistance in Mycobacterium tuberculosis is essential for efficient treatment and control of this pathogen. The amplification refractory mutation system (ARMS) was used to detect mutations in the rifampin resistance-determining region of the rpoB gene. A total of 39 rifampin-resistant M. tuberculosis isolates in Shanghai were analyzed by this assay, resulting in 92.3% sensitivity (36 of 39) and 87.2% concordance (34 of 39) relative to DNA sequencing, by which 41 mutations of 11 different types, including 9 point mutations and 2 deletions, were identified in the rpoB gene. The most frequent mutations were those associated with codon 531 (21 of 39 [53.8%]) and codon 526 (9 of 39 [23.1%]). The results suggest that the ARMS assay is rapid and simple to implement and could be performed for detection of rifampin resistance in M. tuberculosis to complement conventional culture-based methods.Tuberculosis (TB), though curable, still remains a major public health concern worldwide. According to the report of the World Health Organization, about one-third of the world's population (1.86 billion people) are infected with Mycobacterium tuberculosis and are at risk of having the infection develop into clinical TB. Approximately 8 million new cases occur each year, resulting in 3 million deaths around the world (15,19). It has been estimated that among 6 million active TB patients at present, the disease causes 250,000 deaths every year in China (28). Furthermore, control of TB has been further complicated by the emergence of multidrug-resistant (MDR) M. tuberculosis strains and the human immunodeficiency virus epidemic (4, 9).Rifampin, introduced in 1971, has proven to be one of the most potent antituberculosis agents (2). Rifampin is an effective bactericidal against M. tuberculosis, interacting with DNAdependent RNA polymerase to inhibit transcription and elongation of RNA (11,12), and the use of this drug has greatly shortened the duration of chemotherapy. The molecular mechanism of rifampin resistance in M. tuberculosis was first characterized in 1993 (21). Ninety-six percent of rifampin-resistant (Rif r ) M. tuberculosis strains possess genetic alterations within an 81-bp rifampin resistance-determining region (RRDR) in the rpoB gene (16, 18), corresponding to codons 507 to533 (Escherichia coli numbering system). In addition, rifampin resistance can be assumed to be a surrogate marker for MDR TB, since more than 90% of Rif r isolates are also isoniazid resistant (6).Early diagnosis of TB and rapid detection of rifampin resistance are essential for efficient treatment and control of M. tuberculosis. However, culture-based methods for detection of M. tuberculosis infection and testing of drug susceptibility usually take more than 1 month. Although several different genotypic methods, such as PCR single-strand conformational length polymorphism (21, 22), dideoxy fingerprinting (7), heteroduplex analysis (26), and DNA sequencing (13), have been used for analysis of rpoB gene muations associated with r...

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

“…They reported the most frequent mutations in RRDR of rpo B gene are in codon 531 followed by codon 526 and codon 516 [13][14][15][16][17]. However, some of the previous researchers [17] also reported codon 526 as the most common site of mutation leading to rifampicin resistance in MDR-TB cases.…”

Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

“…The working principle behind ARMS-PCR is that oligonucleotides which are complementary to a wild type DNA sequence will only function as primers in PCR under optimized conditions, however, the mutated DNA sequence won't get amplified (Fan et al, 2003). The protocol has been followed for the detection of several genetic polymorphisms including 1-antitrypsin deficiency (Newton et al, 1989), CFTR gene mutation (Ferrie et al, 1992), apolipoprotein E genotypes (Donohoe et al, 1999), and Kras mutation (Carpenter et al, 1996).…”

“…The protocol has been followed for the detection of several genetic polymorphisms including 1-antitrypsin deficiency (Newton et al, 1989), CFTR gene mutation (Ferrie et al, 1992), apolipoprotein E genotypes (Donohoe et al, 1999), and Kras mutation (Carpenter et al, 1996). With respect to its limitation, ARMS PCR can be used for the detection of the mutation only but not its nature, hence cannot substitute for the results obtained through DNA sequencing (Fan et al, 2003).…”

“…The primers used in this study were designed as described by Fan et al(2003).The rationale of primer designing for ARMS PCR is that a single nucleotide mismatch at the 3'-OH extremity of the annealed forward primer renders Taq DNA polymerase unable to extend the primer in the PCR under appropriate conditions. Thus, the absence of the specific PCR product, with a positive result for the internal control, reveals a deviation from the wild-type DNA sequence.…”

“…Thus, the absence of the specific PCR product, with a positive result for the internal control, reveals a deviation from the wild-type DNA sequence. An additional deliberate mismatch (shown in Table 2 as bold and underlined base) adjacent to the 3′-OH terminus of the ARMS primer was introduced in order to enhance discrimination between normal and mutant alleles (Fan et al, 2003).…”