Molecular Typing of Brucella melitensis and B. abortus from Human Blood Samples Using PCR-RFLP Method

Abstract

Background:Brucella is an intracellular parasite of the disease brucellosis throughout the world. Although several molecular typing methods are introduced to find DNA polymorphism that is able to identify Brucella species and biovars, but among these methods, detection of polymorphisms by PCR-RFLP has several advantages including the easy implementation, interpretation and the ease of use for large quantities of samples. Objectives: In the current study, the technique was used for molecular typing of Brucella abortus and B. melitensis that was isolated from human blood samples. Materials and Methods: Blood samples of 160 patients were transferred to Kerman clinical centers with chief complain of acute brucellosis and showed high blood serum level (about 1.80). Their DNAs were extracted by Phenol chloroform method, and the PCR was optimized by using the fragments of designed primers for omp2a and omp2b. Therefore PCR products were restricted by restriction endonuclease as PstI and Hinf1. Finally they were electrophoresed for analyzing the digestion results on agarose gels (2%). Results: In 160 blood samples that were studied with PCR technique, 52 cases obtained bands of 1100 bp for omp2a locus and 1200 bp for omp2b locus from within 52 positive samples by PCR-RFLP method 25 cases (48%) were positive out of which 56% were B. melitensis biovar1 and 44% were B. abortus biovars of 3, 5, 6 or 9. Conclusions:The results of this study showed that PCR-RFLP technique was a fast and applicable method especially for separation, detection and differentiation between species of B. melitensis and B.abortus biovars in blood sample. Also the presented data showed that B. melitensis biovar 1 was the prevalence biovar.

Molecular Typing of Brucella Species Isolated from Humans and Animals Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Technique

“…These results indicated that PCR-RFLP of omp2a gene was not able to accurately discriminate human and animal B. melitensis biovars from each other and from B. abortus. Also, comparable results were reported by Mirnejad et al and Pishva et al (6,10).…”

“…and omp2b fragments and several restriction enzymes were used in various studies (10,25). In the present study, similar to other studies in Iran and different parts ofthe world, amplification of omp2a gene from clinical and animal isolates showed different sizes (50 to 500 bp) (1,26).…”

“…Homology of DNA for all species in the genus of Brucella is more than 90% and probable new biovars and strains of Brucella enter the area. Consequently, investigations about the epidemiology of brucellosis, pathogenicity, and ecological differences between species and biovars of Brucella may help choose the correct vaccine, plan epidemiological studies, and control and eradicate the condition (10,11). Molecular techniques have shown accurate typing of Brucella spp.…”

Typing and whole-genome sequencing of Brucella isolated from clinical samples in Lebanon

“…PCR was performed on sequenced isolates in a 50 ul final reaction volume containing 1x PCR buffer (Thermoscientific, USA), 2 mM MgCl 2 (Thermoscientific, USA), 0.15 mM dNTPs (Thermo Fisher Scientific), 2.5 U Taq polymerase (Thermoscientific, USA), 20 pmol of primers 2aA (5'-GGCTATTCAAAATTCTGGCG-3') and 2aB (5'-ATCGATTCTCACGCTTTCGT-3') and 0.5 ug of template DNA (Cloeckaert et al, 1995;Mirnejad et al, 2013). Cycling conditions were as follows: pre-denaturation step for 5 min at 94 ºC, followed by 35 cycles of denaturation at 94 ºC for 60 s, annealing at 50 ºC for 120 s, and extension at 72 ºC for 180 s, and finally an extension step at 72 ºC for 7 min.…”

“…Conducting the omp31 PCR assay on all the isolates revealed that all carried the gene, thus confirming that they don't belong to B. abortus. In addition to that, the obtained restriction digestion pattern, P1, using HaeIII enzyme ruled out B. suis biovar 2. omp2a is another candidate and target for PCR-RFLP analysis and differentiation of species and biovars of Brucella (Cloeckaert et al, 1995;Mirnejad et al, 2013). It is considered as an immunogenic antigen in addition to being a virulence factor (Cloeckaert et al, 1996).…”