Iranian South Medical Journal (Aug 2014)
Improvement of the Molecular diagnosis of Cryptococcus neoformans using Internal Control (IC)
Abstract
Background: Lacking of internal control in majority of the PCR realated searchesis one of the most important items yet. Using sensitive and specialized laboratory techniques such as PCR is necessary to cryptococosis diagnosis. Different methods to detect this pathogen exist. Generally, culturing based methods are time consuming and have low sensitivity and need enough experience and equipments to data analyzing. Further painting based methods have no sufficient sensitivity as well. As a result, molecular techniques such as PCR are more appropriate to diagnosis purposes, but different data gathered from non standard tests supposed to be one of the deficiencies of this powerful molecular technique. This aim of this study was to design and produce the plasmid amplification internal control (IAC) to identify the restrictors in Cryptococcus neoformans PCR test and its future applications in the routine diagnostic laboratories. Materials and methods: In this study to produce internal control, first the special PCR primers based on Gene target 16SrRNA for optimized molecular detection and then sensitivity and character were identified. Then, the composite primers for IAC C. neoformans was designed, replicated and clonized as well. The replicated IAC C. neoformans was attached to pTZ57R and transformed and colonized in E.coli JM107. The minimum IC number of each PCR reaction was studied using dilution and PCR reaction spectrum with IC. Results: The size of C. neoformans diagnostic product with its special primers was 415 bp and IAC C. neoformans amplicon is 661 bp, which have desired deference in the size (246 bp). The minimum IC number was identified 1000 in each reaction. The min/max sensitivity of PCR test with IC for C. neoformans DNA was identified between 100 particle to 3 million yeasts. There was no unwanted product in the character test with different agents. Conclusion: In spite of high rate and exclusivity of PCR technique, one of the main difficulties of this exacting method is the false positive and negative results which occur because of different reasons which may led to decrease its performance. Using an internal control for molecular diagnosis of Cryptococcus neoformans as inside controlling systemcan detect these errors.
