Sensitivity Of Duplex Real Time Pcr

(Tortora, Funke & Case, 2013). Another mycobacterial species, Mycobacterium bovis, is a pathogen mainly of cattle. M.bovis is the cause of bovine tuberculosis, which is transmitted to humans via contaminated milk or food (Tortora, Funke & Case, 2013). It is very unlikely to see M. bovis transmitted from person to person. It will impinge bones causing a hunchback deformation of the spine; it also will affect the lymphatic system. In the latent stage, the patient is asymptomatic due to TB is inactive. Symptoms may appear weeks or even years later after acquiring the infection. In the active stage symptoms will include a cough that may last three weeks or more, expectorating blood, erratic weight loss, more tiredness than usual, fever, Night sweats and chills. Active TB is indicative to the spread of the infection; which happens when the bacteria advances from person to person from unhindered microscopic droplets dispensed into the air. Two of the most-common test used to the diagnosis tuberculosis is a simple skin test and chest x-ray. The skin test is done by injecting a minute amount of PPD under a patient's skin creating a welt on the forearm. The results are read in 48-72 hours by a health professional; they will check for swelling at injection site. If the PPD test reads false positive then, a chest x-ray will be ordered. Chest x-rays are

Mycobacterium tuberculosis is a pathogen, which its physiology is directly linked to features of tuberculosis that it causes. The crucial feature for a mycobacteria’s survival is its unique cell wall structure. The insoluble cell wall core of MTB is formed by a large variety of lipid-containing molecules, such as mycolic acid, that are covalently attached (6). This hydrophobic cell wall provides a physical protection from the host immune response and serves as a barrier against many toxic insults (2). Further, the complex MTB cell wall is impermeable to both hydrophobic and hydrophilic molecules, resulting in inherent resistance of MTB to most common antibiotics (8). Lipoarabinomannan is an antigen on the outside of the organism. This antigen is another important component of the cell wall because it inhibit the fusion of Mycobacterium-containing phagosomes with lysosomal compartments (4). Lipoarabinomannan hinders the fusion of phagosome with lysosome by impairing Ca2+/calmodulin pathway and inactivates macrophages (8). Therefore, this cell-surface component of MTB is able to facilitate the survival of mycrobacteria within macrophages (8). Also, MTB is able to survive the harsh environment of the host tissues by utilizing any available

Buckinghamshire, UK) were used within each supplied tube. Each bead contains the desired ingredients needed for the successful PCR reaction, stabilizers, bovine serum albumin to increase overall yield, four deoxynucleotides triphosphates (dNPT), adenine, guanine, cytosine, tyramine ATP, approximately 2.5 units of puReTaq DNA polymerase and a reaction buffer. When reconstituted to the final volume of 22µl, the final volume of each dNTP is 176µM in 8.8mM of Tris-HCL which at room temperature has a pH of 9.0. Finally, 44mM of potassium chloride and 0.66mM of magnesium chloride. All PCR was preformed within a fume cupboard (Bassaire 06HB) to minimise the risk of either moisture or genomic

Mycobacteria are rod-shaped bacteria which require oxygen for growth. Each species has an acid-fast staining property during some stage of its growth cycle. It has thick, waxy, outer coating which can lead them to thrive in aquatic environments. For some time, scientists have known of bacteria that are similar to Mycobacterium tuberculosis, but that grow and act differently. When tuberculosis was a much more widespread problem and microbiology was much less able to tell the difference between similar microbes, these atypical mycobacteria were ignored. Today, they have been classified more precisely as members of the same species and called atypical (or nontuberculosis) mycobacteria. Although the medical profession has known about these atypical

Urinalysis: Diagnosis of genitourinary TB is established with Koch’s bacillus demonstration in urine. The presence of dysuria, sterile pyuria, and haematuria are highly suggestive for diagnosis. In TB infection, bacterial overlap may occur, so positive bacterial culture should not abolish the possibility of urinary TB. Traditionally TB diagnosis is established by M. tuberculosis isolation: direct microscopic examination for the detection of acid-fast bacilli (AFB) colouring in Ziehl-Neelsen is the fastest method for identification of mycobacteria providing,

Being able to determine the amount of DNA present in an organism has been successfully done using qPCR. In this experiment, qPCR was used to identify how much fungi could infect mutant and wildtype lines of A. thaliana. Also, qPCR was used to amplify the 16s rRNA ITS (internal transcribed spacer) of an unknown fungal pathogen. Other ways qPCR has been used in recent years include the use of qPCR to quantify copy number variants in the HER-2 gene which is a proto-oncogene. This method was particularly employed in the formalin-fixed-parafilm-embedded tissues - due to the fact that their DNA is usually broken into smaller pieces – to gain accurate results. This study concluded that qPCR produced similar results with already existing

In such necrotic granulomas, infected AMs undergoes cell lysis and release slowly-replicating or non-replicating mycobacteria in to caseous center. These necrotic granulomas with caseous center may create a suitable niche for slow growing reservoir mycobacteria (Seiler et al., 2003; Fenhalls et al., 2002; Ryan et al., 2010; Driver et al., 2012; Hoff et al., 2010). Sterilization of such necrotic granulomas containing slow growing or non-replicating mycobacteria is extremely

[4-5] Though these species are extremely related there relevance to certain disease manifestation is dissimilar. In order to resolve these species modern techniques must be implemented. Probes have been designed to target and amplify uniquely identifiable regions on the genomes of these two species. [5] By using Polymerase chain reactions (PCR) the ratio of M. avium and M. intracelluare can be ascertained for each manifestation of disease. Other members of Mycobacteria can be isolated and identified in the MAC including M. chimaera and M. colombiense. [2]

Myobacterium tuberculosis is the cause of tuberculosis in most patients. It is a long and thin acid-fast rod. It is a strict aerobe and is not referred to as a gram-positive or gram-negative because its acid-fast nature is much more relevant in a clinical setting. It grows very slowly; with a generation time of 15 to 20 hours, a period of up to 6 weeks is required for colonies to appear in culture.

A new rapid test that overcomes many of the current operational difficulties was recommended for use by WHO in December 2010: the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA). The test is based on a real-time heminested PCR test which detects the presence of M. tuberculosis complex bacilli.15By using 5 molecular beacons which span the rpoB gene 81-bp rifampin resistance-determining region (RRDR) in Mycobacterium.16The Xpert MTB/RIF assay represents a paradigm shift in the diagnosis of TB and drug-resistant TB by simultaneously detecting MTB and rifampicin resistance-conferring mutations in mycobacterium in a closed system suitable for use outside conventional laboratory

M. tuberculosis was first described by Robert Koch in the year 1882 as the “tubercle bacillus”. The bacterium belongs to a special group of microorganisms that contain a thick, waxy, lipid rich layer of mycolic acid on their cell surface. The mycolic acid makes this bacterium tolerant to a number of antimicrobials and is one of its major virulence factors. Due to the presence of the mycolic acid layer, it is very difficult to stain Mycobacterium with conventional Gram staining. Thus, a special staining technique called acid-fast staining or Ziehl-Neelsen staining is used to stain the TB pathogen. Although, M. tuberculosis do not stain well during Gram staining, they are often described as acid-fast Gram positive bacilli. They are called Gram positive as they do not have an outer cell membrane like Gram negative bacteria. M. tuberculosis are aerobic, non-motile, intracellular pathogens that most commonly infect the mammalian pulmonary system. It has a very slow generation time as it undergoes one division every 15 to 20 hours.

Previous work with DNA based technologies to accurately detect and identify human pathogens has been demonstrated. Furthermore, three methods have been utilized: 1) amplification of one or more universal genes (16S rRNA and 23S rRNA) through PCR, 2) amplification of pathogen-specific markers (toxins, virulence factors) using multi-plex PCR and 3) amplification of random DNA fragments (Kim et al., 2008). Kim et al. (2008) state that the first two methods is flawed due to the limited number of probes utilized and that the amplification of universal genes would not discriminate below species level due to the fact that these genes are highly conserved within the genus. On the other hand, Kim et al. (2008) reported that

The sensitivity of the Xpert MTB/RIF was considerably better than the sensitivity of the Cobas TaqMan MTB assay. The difference in PCR technology could account for the substantial difference in sensitivity. The Xpert MTB/RIF uses heminested real-time PCR technology whereas the Cobas Taqman MTB assay uses simple real-time PCR technology. The Xpert MTB/RIF assay system also looks to be less susceptible to cross-contamination than other PCR based methods. Cross-contamination could give false positive results (Blakemore, et al., 2010). A major benefit of these molecular methods is that they both provided results in less than 2 hours and required very little handling. The Xpert MTB/RIF assay system in particular requires very little handling by laboratory staff as extraction, amplification and detection occur in a single-use cartridge, which ensure minimal contamination of the specimen. There is a greater chance for contamination in the Cobas TaqMan MBT assay as the specimens are extracted manually. Another benefit of the Xpert MTB/RIF kit is that samples can be processed individually as they are needed, whereas the Cobas TaqMan MTB assay requires at least 10 samples and 2 controls to be processed at a time in order not to waste reagents. These molecular methods represent great progress made in methods for detecting M. tuberculosis. Reference culture methods can talk up to 6 weeks to get a final diagnosis whereas the real-time PCR and

PCR reaction was carried out in a 25 μl reaction mixture under standard condition, contained 1x buffer, 0.2 mM of four dNTPS, 1.5mM mgcl2, 1 pM of forward (sequence) and reverse (sequence) primers and 50ng of genomic DNA. Initial denaturation at 95˚C for 4 min was followed by denaturing at 94˚C for 30 seconds, annealing at 60˚C for 30 seconds and extension at 72˚C for 30 seconds for 32 cycles. The final extension was 8 min. The amplicons were run on an ABI 3130x (Applied Biosystems, Foster City, California,

One-forth of this world’s people are infected with Mycobacterium tuberculosis. It is the reason of virtually millions of deaths all over the world every year. This ratio is more than the deaths caused by any other pathogen. From the start of the twentieth century, tuberculosis has become a relatively uncommon disease instead of the most common reason of deaths worldwide [1-3]. The incidence of tuberculosis has waned in the developed countries. The World Health Organization reports that more than ten million cases and two to three million deaths occur annually due to tuberculosis [5]. By another estimate, it is said that at least one billion persons are infected with M. tuberculosis worldwide [6].