Hellow my fellows
Iam working on Real time PCR technique for detection the gene who is responsiable on Tuberculosis as well as the AFB stain so i hope that any one have worked on the same project that can assist me by sending articles or researches that might be useful to complete it .
Thanks
Hammad Shihab
Assistant lecturer
College of education for girls
Mosul University
Daer Hammad!
You can look at this article:
Article Detection of Tuberculosis (TB) using Gold Standard Method, D...
Acid Fast Bacilli (AFB) Staining There are two commonly used procedures for staining, which are the direct microscopy and fluorescent microscopy. Direct microscopy uses carbolfuchsin method and is divided into two; Ziehl Neelsen and Kinyoun methods. According to Tankeshwar [15], both methods are still widely used in developing country. For fluorescent microscopy, fluorochrome procedure using auramine-O or auramine-rhodamine dyes is commonly used [16]. It is recommended by CDC to use fluorochrome staining rather than direct microscopy to detect AFB in primary patient specimens [17].
Real-time or Quantitative Polymerase Chain Reaction (qPCR) Recently, real-time PCR or qPCR has been widely used because of its excellence performance to overcome the limitation in conventional PCR method. The qPCR is believed to be more rapid than conventional PCR with excellent reproducibility that only requires less than one hour in diagnosing MTB [32] (after DNA extraction). Despite its short experimental time, qPCR remains as low risk of sample contamination and offers the possibility of quantifying a bacterial load [33]. According to Haron and friends [34] for qPCR optimisation, the sequence of probe can be modified from TB probe of IS6110 gene with quenchers including FAMGAACGGCTGATGACC AAACT-BHQ-1. A 5` end of the probe was labelled with 6-carboxy-fluorescence-dye (FAM) and a 3` end was labelled with non-fluorescence dye (BHQ-1). The DNA from bacterial isolates and clinical samples were subjected to Real-time PCR using the mixture of TB primers and TB probe. The reaction mixture consists of template DNA, TaqMan MasterMix, primers and probe. Analysis was carried out at 60°C for 1 min. The cycling parameters for Absolute Quantification were done at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 1 min. The data were collected during annealing steps. It was reported in the previous study by Park and colleagues [35] that qPCR has the sensitivity and specificity of 83.7 % and 96.2 %, respectively, while sensitivity and specificity of smear microscopy are only 56.8 % and 99.6 %, respectively. Previous study by Nagdev and colleagues [33] also reported that the overall accuracy of qPCR was found higher than conventional PCR. Thus, it can be concluded that molecular diagnosis of TB can be improved by qPCR.
Dear Alexandr Chernov, Dr Victor Hugo Dias ([email protected]) at UFRJ defended his PhD yesterday and in it presented a MPS NGS method for detecting potentially dangerous bacteria for human health. In his thesis 52 primer sets. With colleagues he has also published a paper in 2020 which will give you what you are asking for. Best of luck
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