Molecular Identification of Nocardia Strains from the Soil by hsp65 Gene: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (RFLP) and Analysis of Sequence 16S rRNA Gene

Background: Nocardia species are nocardiosis agents particularly in immune disorder patients with the clinical manifestations including fatigue, malaise, weight loss, cough, and dyspnoea. Due to time - consuming bacterial culture and insufficient phenotypic tests alone, rapid molecular assays for the diagnosis of Nocardia are essential. The primary objective of this study was the analysis of the polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP) and PCR sequencing of 16S rRNA gene for the identification and differentiation of Nocardia species. Methods: 32 Nocardia isolates were identified by single digestion of 441 - nt fragments of the 65 - heat - shock protein gene with endonucleases BstEII, MspI, and HinfI and sequencing of 16S rRNA gene. These isolates were collected from the soil of various geographical regions of Iran by paraffin baiting technique in our previous study. Results: Some of the isolates had new patterns that were not identified by PCR - RFLP of hsp65 gene in literature; therefore, they were identified by sequence analysis of 16S rRNA gene. Six strains of Nocardia with new DNA banding patterns in PCR - RFLP of hsp65 (A and B) could not be identified by their hsp65 restriction - endonucleases fragment patterns. Strain En49 after analysis of sequencing data was recognized as Nocardia coubleae, which its PCR - RFLP profile of hsp65 has not been reported in the literature so far. Conclusions: The PCR - RFLP - hsp65 is faster than other sequencing-based techniques, but species accurate identification in strains with new PCR - RFLP profiles is not possible. The 16S rRNA gene sequencing is a suitable method to determine the percent similarity and phylogenetic relationships of Nocardia species.