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K.L. Therese, J. Therese,P.Deepa ,R.Pusphalatha,H.N.Madhavan; Evaluation of Genotypic and Phenotypic Identification Methods for Speciation of Ocular Isolates of Mycobacteria . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1427.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the ocular mycobacterial isolates to species level rapidly by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) technique using three sets of primers coding for two different genes of mycobacteria and to evaluate the techniques with the conventional standard biochemical tests . Methods: :Eighteen ocular mycobacterial isolates (7 corneal scrapings, 4 Fine needle aspiration biopsy (FNAB) from sub retinal mass, 2 cannalicular pus , 1 scleral iris mass , 3 intraocular fluids, and 1 intraocular lens(IOL) from 14 patients were included in the study. PCR-RFLP technique using three sets of primers (setI-coding for 16-23 ribosomal DNA interspacer sequences Sets II & III-coding for HSP 65 gene) were standardised for specificity and sensitivity and applied on all 18 isolates of mycobacteria.The amplified products of were digested with restriction enzymes,Hae III,Msp I and Bst XI , primer set I Xho I and Bst NI with Primer set II, and Hae III, and Bst E11 with primer set III.The identification of the mycobacterial isolates were carried out by conventional biochemical tests, including substrate utilzation tests. Results: The primer set I could differentiate the 18 isolates into 7 slow growers (325-370 bp) and 11 rapid growers (420-520 bp)of mycobacteria. The primer set II and III resulted in 1380 bp and 439 bp amplified products respectively for all mycobacterial isolates. Further based on the RFLP pattern all the 7 slow growers (4 FNAB from sub retinal mass,1 corneal scraping 1 scleral iris mass, 1 cannalicular pus) were identified as Mycobacterium tuberculosis and among the 11 rapid growers 10 were identified as Mycobacterium chelonae and 1 as Mycobacterium fortuitum Conclusions: To identify ocular mycobacterial isolates to species level, the conventional biochemical techniques required minimum 3 weeks for rapid growers, and 4 weeks for slow growers whereas the PCR-RFLP techniques required only 8-10 hours time.Among the 3 PCR-RFLP techniques the primer set III coding for 65KD gene was considered as the most suitable technique for rapid and accurate identification of mycobacteria to species level.
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