Subsequent to the introduction of commercially available hybridization assays, commercially available and in house–developed nucleic acid amplification tests were used successfully for early identification of M. tuberculosis complex grown in liquid cultures. Currently, PCR-based sequencing for mycobacterial identification consists of PCR amplification of mycobacterial DNA with genus specific primers and sequencing of the amplicons. The organism is identified by comparison of the nucleotide sequence with reference sequences. The most reliable sequence for identification of mycobacteria is the approximately 1500 bp 16S rRNA gene. However, only a 600 bp sequence at the 5′ end is required for identification. The sequence homogeneity in the M. tuberculosis complex prevents the use of this sequence to differentiate these species. This region contains both conserved and variable regions, which makes it an ideal target for identification purposes.

Despite the accuracy of PCR-based sequencing to identify mycobacteria, problems remain: the sequences in some databases are not accurate; no present consensus exists as to the quantitative definition of a genus or species based on 16S rRNA gene sequence data; and procedures are not standardized. In addition, the 16S rRNA 5′ region contains two hypervariable regions, A and B. The A region provides the signature sequences for species identification. However, M. chelonae and M. abscessus both require additional sequencing, because the A and B regions are identical and the 3′ end of the 16srRNA contains a 4-bp sequence difference.

Several other genes have also been used to identify mycobacterial species, including the 23S rRNA, ITS 1, hsp65, rpoB, and gyrB gene. The 23S rRNA sequence is 3100 bp in length, which limits accurate sequencing. ITS 1 is a spacer sequence located between the 16S and 23S rRNA genes. This sequence, which is only 200 to 330 bp, is more easily analyzed. The limitation of this sequence is that it is not a genus-specific sequence; therefore, results may be affected by contaminating bacteria. The 65 kDa heat shock protein, also referred to as the groEL2 gene, is a 440-bp fragment that can be amplified and analyzed with restriction digestion, followed by agarose electrophoresis. The hsp65 is highly conserved but contains a greater variation in polymorphisms than the 16S RNA, particularly in a 441-bp region referred to as the “Telanti fragment.” This allows for differentiation of Mycobacterium species based on the variation in restriction fragment length polymorphisms (RFLPs). Repetitive sequence–based PCR, Diversilab (Biomérieux, Durham, N.C.), demonstrates better species discrimination than RFLP. In addition, a commercially available system in which the 16S to 23S rRNA spacer region of mycobacterial species (INNO-LiPA Mycobacteria; Innogenetics, Ghent, Belgium) has been successfully used to directly detect and identify several of the most clinically relevant mycobacterial species in aliquots of positive liquid culture. However, caution should be used in interpretation of results, because some cross reactivity has occurred with closely related species.

Another commercial system, GenoType Mycobacterium (Hain Lifescience GmbH, Nehrin, Germany), which uses a similar format, has additional probes from M. celatum, M. malmoense, M. peregrinum, M. phlei, and two subgroups of M. fortuitum, in addition to a supplemental kit that allows for 16 additional mycobacterial species. Yet another commercial system, MicroSeq500 16S rRNA (Applied Biosystems, Foster City, California), sequences a 500-bp region and uses a comparative database for species identification.

The rpoB gene encodes the beta-subunit in the organ ism’s RNA polymerase. Mutations in this gene confer rifampin resistance to M. tuberculosis. Different regions in this gene have been used to identify rapid-growing iso lates, but little data are available for the slow-growing species. Finally, the gyrB gene encodes the beta-subunit in the organism’s topoisomerase II. Several single nucleotide polymorphisms have been identified in this gene that are useful in distinguishing species in the M. tuberculosis complex. After amplification, identification and differentiation of species requires restriction analysis and gel electrophoresis.

Additional molecular techniques, such as conventional and real-time PCR, have been used to detect M. tuberculosis directly in clinical specimens. For example, the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic Systems, Branchburg, New Jersey) uses PCR to detect M. tuberculosis directly in respiratory specimens. The Amplified Mycobacterium tuberculosis Direct Test (AMTD; Gen-Probe, San Diego, California) is based on ribosomal RNA amplification. The Roche assay currently is approved by the FDA for use only on acid-fast, smear positive specimens, because numerous studies have demonstrated less than optimum sensitivity on smear-negative specimens. Because of subsequent kit modifications that improved sensitivity, Gen-Probe’s assay is approved on both smear-positive and smear-negative specimens. Ribosomal RNA is released from the mycobacteria by means of a lysing agent, sonication, and heat. The specific DNA probe is allowed to react with the extracted rRNA to form a stable DNA-RNA hybrid. Any nonhybridized DNA–acridinium ester probes are chemically degraded. When an alkaline hydrogen peroxide solution is added to elicit chemiluminescence, only the hybrid bound acridinium ester is available to emit light; the amount of light emitted is directly related to the amount of hybridized probe. The light produced is measured on a chemiluminometer. Numerous laboratories have incorporated these tests into their routine procedures.

The Amplicor test (Roche Diagnostic Systems), which uses TaqMan technology and is a real-time PCR test, has received FDA approval for smear-positive respiratory specimens from patients suspected of havingtuberculo sis. These tests are limited in the number of species they are able to identify. Clinical laboratories have developed their own PCR assays to detect M. tuberculosis directly in clinical specimens.

Line probe assays (DNA strip assays) involve PCR amplification coupled with a reverse hybridization step. The target sequence is amplified using biotinylated primers. The amplicon is then hybridized to membrane immobilized, sequence-specific probes for each species. The membrane is developed using an enzyme-mediated reaction and color indicator to analyze the banding pattern. Banding patterns are species specific based on the immobilized probe map on the membrane. A commercially available line probe assay (GenoType MTBC; Hain Lifescience, Nehren, Germany) enables the identification of M. tuberculosis complex organisms at the species level using the 23S rRNA.

In addition to assays developed in-house and the Genotype MTBC, five non-FDA-approved commercial amplification tests are widely used outside the United States. The Artus M. tuberculosis PCR kit (Qiagen GmbH, Hilden/ Hamburg, Germany) assay uses real-time PCR for amplification of the 16S rRNA gene; the ProbeTec Direct TB energy transfer system (Becton Dickinson, Sparks, Mary land) uses strand displacement amplification technology; the RealArt M. tuberculosis TM PCR reagents (Abbott Laboratories, Abbott Park, Illinois) is a real-time PCR assay using the ABI Prism 7000 system; and the Loop mediated isothermal amplification test (Eiken Chemical, Tokyo) uses isothermal amplification and UV light detection. These systems have sensitivities and specificities comparable to those of the FDA-approved amplification assays. The GeneXpert system (Cepheid, Sunnyvale, California), which is used for real-time PCR detection of M. tuberculosis complex and resistance to rifampin, uses amplification of the rpoB gene previously discussed in this section.

Currently no molecular assays are available for direct detection of nontuberculous mycobacteria. In 2004, the Centers for Disease Control established a national tuberculosis genotyping system. Details and updates are avail able at http://www.cdc.gov/tb/programs/default.htm.

DNA Microarrays. DNA microarrays are also attractive for rapid examination of large numbers of DNA sequences by a single hybridization step. This approach has been used to simultaneously identify mycobacterial species and detect mutations that confer rifampin resistance in mycobacteria. Fluorescent-labeled PCR amplicons generated from bacterial colonies are hybridized to a DNA array containing nucleotide probes. The bound amplicons emit a fluorescent signal that is detected with a scanner. With this approach, 82 unique 16S rRNA sequences allow for differentiation of 54 mycobacterial species and 51 sequences that contain unique rpoB gene mutations (mutations that confer resistance to rifampin).

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم شؤون المعارف ينظم دورة متقدمة في تحقيق المخطوطات لملاكاته

قسم الشؤون الفكرية يصدر العدد العشرين من مجلة دراسات إفريقية

المجمَع العلمي يكرم الأساتذة المشاركين في مشروع الدورات القرآنية الصيفية في كربلاء

العتبة العباسية المقدسة تختتم فعاليات البرنامج المركزي الأسبوعي لمنتسبيها

المزيد

الآخبار الصحية

هل من الآمن شرب القهوة على معدة فارغة؟

تحذير: مرض السكري يسرق طاقة قلبك

حقيقة صادمة.. لماذا يجب أن تتوقف عن شراء الجزر الصغير؟

بعد عمر الستين.. توقيت وجبة الإفطار مؤشر على صحتك

المزيد

الآخبار التكنلوجية

زلزال أفغانستان يحصد أكثر من ألف قتيل.. ويدمر آلاف المنازل !

ناجٍٍ وحيد من انزلاق أرضي.. دمّر قرية سودانية بأكملها !

نهر الفرات.. مورد مائي بين الندرة والتقاسم

ما السر وراء مشية إيلون ماسك الغريبة والسريعة ؟

المزيد

مواضيع ذات صلة

Conventional Phenotypic Tests for Mycobacterial Infections

Acid-Fast Stains for Mycobacterial Infections

Vibrio Parahaemolyticus and Vibrio vulnificus

Vibrio Cholerae

Burkholderia cepacia Complex

Burkholderia pseudomallei and Burkholderia mallei

Rapidly Growing Nontuberculous mycobacteria (RGM)

Slow-growing Nontuberculous Mycobacteria

Mycobacterium Tuberculosis Complex

Anaerobic Gram-Positive and Gram-Negative Cocci

The Salmonellae

The shigellae