PCR - RFLP patterns for the differentiation of the Fusarium species in virtue of ITS rDNA

Background and Purpose: The Fusarium species are among the most important fungi in the medical, veterinary and agricultural fields. Materials and Methods: In the present study, 172 strains of these fungi have been analyzed. The high molecular weight DNAs were extracted from 23 reference strains as well as from 149 isolated Fusarium species. Using the designed nucleotide primers from rDNA of Fusarium species, PCR analysis was performed for the amplification of ITS regions. Afterwards, the location of the effective endonuclease enzymes has been evaluated within approximately 930 bp of rDNA sequence. Results: Through the selected enzymes including; HhaI, MspI, TaqI and FaqI, the mentioned Fusarium species have been divided into 33 groups. The first three enzymes were able to classify Fusarium species into 23 groups of which 19 groups included one member, one group included two members and three groups included three members of the Fusarium species. This study also revealed the possibility in the identification of F. semitectum, F. solani complex, F. pseudograminearum, F. nisikadoi, F. coeruleum and F. acuminatum species by one unique enzyme. In addition, our study indicated the ability of the differentiation of F. Compactum from F. equiseti. Conclusion: As Compared to previous studies with more endonuclease enzymes and with limited in identifications, the ITS-RFLP patterns reported here an attempted to evaluate most of the Fusarium species successfully

Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella

It is of great importance to quickly and accurately detect Brucella abortus and Brucella melitensis from clinical and non-clinical samples because of their high prevalence and high risk in causing brucellosis, a life-threating infectious disease affecting both humans and animals. The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared. The mean range cycles threshold±standard deviation (CT±SD) for the amplified samples was 15.39-25.15 by real-time PCR. The melting peak range (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak height. The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing Brucella quickly and precisely. Quick recognition of Brucella species could decrease its prevalence among humans and animals and mitigate economic loss. © 2017 2017 Walter de Gruyter GmbH, Berlin/Boston

Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella

It is of great importance to quickly and accurately detect Brucella abortus and Brucella melitensis from clinical and non-clinical samples because of their high prevalence and high risk in causing brucellosis, a life-threating infectious disease affecting both humans and animals. The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared. The mean range cycles threshold±standard deviation (CT±SD) for the amplified samples was 15.39-25.15 by real-time PCR. The melting peak range (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak height. The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing Brucella quickly and precisely. Quick recognition of Brucella species could decrease its prevalence among humans and animals and mitigate economic loss. © 2017 2017 Walter de Gruyter GmbH, Berlin/Boston

Species-specific PCR for the diagnosis and determination of antibiotic susceptibilities of brucella strains Isolated from Tehran, Iran

Background: Brucellosis is an endemic zoonotic disease in the Middle East. This study intended to design a uniplex PCR assay for the detection and differentiation of Brucella at the species level and determining the antibiotic susceptibility pattern of Brucella in Iran. Methods: Sixty-eight Brucella specimens (38 animal and 30 human specimens) were analyzed using PCR (using one pair of primers). Antibiotic susceptibility patterns were evaluated and compared using the E-Test and disk diffusion susceptibility test. Tigecycline susceptibility pattern was compared with other antibiotics. Results: Thirty six isolates of B. melitensis, 2 isolates of B. abortus and 1 isolate of B. suis from the 38 animal specimens, 24 isolates of B. melitensis and 6 isolates of B. abortus from the 30 human specimens were differentiated. The MIC50 values of doxycycline for human and animal specimens were 125 and 10 μg/ml, respectively, tigecycline 0.064 μg/ml for human specimens and 0.125μg/ml for animal specimens, and trimethoprim/ sulfamethoxazole and ciprofloxacin 0.065 and 0.125μg/ml, respectively, for both human and animal specimens. The highest MIC50 value of streptomycin in the human specimens was 0.5μg/ml and 1μg/ml for the animal specimens. The greatest resistance shown was to tetracycline and gentamicin, respectively. Conclusion: Uniplex PCR for the detection and differentiation of Brucella at the strain level is faster and less expensive than multiplex PCR, and the antibiotics doxycycline, rifampin, trimethoprim-sulfamethoxazole, ciprofloxacin, and ofloxacin are the most effective antibiotics for treating brucellosis. Resistance to tigecycline is increasing, and we recommend that it be used in a combination regimen. © 2016, Iran J Pathol. All rights reserved