Improvement Of Acid-fast Stain Method For Nocardia And Genotyping Of Nocardia With PCR-RFLP Based RpoB Gene

Objectives:In this study we improved the traditional acid-fast staining method and used rpoB gene as a target gene to establish the standard operating procedure of PCR-RFLP and RFLP standard fingerprint for the rapid identification of Nocardia bacteria,and would like to provide a reliable reference standard for clinical rapid and accurate differential diagnosis of Nocardia disease.Methods:1. To use Gram staining method,acid-fast staining method and use7concentration gradient sulfuric acid as destaining solutions of modified acid-fast staining method to stain Nocardia species respectively,then compare the results of three staining methods comprehensively.2. In this study, a360bp fragment of rpoB gene of Nocardia was used as a target gene for establishing PCR-RFLP. PCR amplification and DNA sequencing of the target gene were performed for detecting all23reference strains of7species of Nocardia. The PCR amplification products from23strains of7species of Nocardia were analysed by DNA sequencing and PCR-RFLP respectively.Results:1. There are three forms of Nocardia under microscope, such as rod-shaped,filamentous and globular.The highest positive rate (100%) can get by use0.125%sulfuric acid or0.25%sulfuric acid as destaining solution, while the lowest positive rate (30.43%) can get by use4%sulfuric acid as destaining solution. Both positive control and negative control can get our expected results when use4%sulfuric acid、2%sulfuric acid、1%sulfuric acid and0.5%sulfuric acid as destaining solution;positive control can acquire positive result, while negative control can acquire false positive result when use0.25%、0.125%and0.063%sulfuric acid was used as destaining solution.2. The results showed that using rpoB gene as a targeted gene,7patterns were obtained when the360bp purified PCR products digested with restriction enzymes HaeⅢ, by which23strains of7species of Nocardia were classified into7kinds respectively; and16patterns were obtained when the360bp purified PCR products digested with restriction enzymes Mspl, by which23strains of7species Nocardia were clssified into16kinds respectively;16kinds of patterns were got with the combination of restriction enzymes HaeⅢ and Mspl. Nocardia nova and Nocardia otitidiscavarium have species specific patterns, Nocardia asteroides has three patterns, Nocardia brasiliensis has four patterns, Nocardia carnea has two patterns and Nocardia transvalensis has two patterns.Conclusions:1. The reformative acid-fast staining method with0.5%sulfuric acid as destaining solution was comfirmed the best staining method for Nocarida based on accurately results of both positive control and negative control, of which the highest detection rate was91.3%.2. The PCR-RFLP method based on rpoB gene has been established successfully by this study, by which16patterns gotten23strains of7species Nocardia are classfied into species. It will provide good basis for clinical Nocardia identification and control of the disease caused by Nocardia.3. The results of species identification showed that all the37clinical strains belong to two Nocardia species, including36Nocardia farcinica and1Nocardiagloberula.